Publications by authors named "Sandra Citi"

49 Publications

The tight junction protein cingulin regulates the vascular response to burn injury in a mouse model.

Microvasc Res 2020 11 30;132:104067. Epub 2020 Aug 30.

Skin and Endothelium Research Division, Department of Dermatology, Medical University Vienna, Vienna, Austria. Electronic address:

Edema formation due to the collapse of physiological barriers and the associated delayed healing process is still a central problem in the treatment of burn injuries. In healthy individuals, tight junctions form a barrier to fluid and small molecules. Cingulin is a cytoplasmic component of tight junctions and is involved in the regulation of the paracellular barrier. Endothelial specific cingulin knock-out mice provide new insight into the influence of tight junction proteins on edema formation and angiogenesis during wound healing. Knock-out mice lacking the head domain of cingulin in endothelial cells (Cgn) were created by breeding Cgn mice with Tie1-cre mice. Using a no-touch hot air jet a burn trauma was induced on the ear of the mouse. Over a period of 12 days microcirculatory parameters such as edema formation, angiogenesis and leukocyte-endothelial interactions were visualized using intravital fluorescence microscopy. At baseline, Cgn mice surprisingly showed significantly less tracer extravasation compared to Cgn littermates, whereas, after burn injury, edema was consistently higher in Cgn mice. Non-perfused area after wounding was increased, but there was no difference in vessel diameters, contraction or dilation of arteries in Cgn mice. Moreover, cingulin knock-out did not cause a difference in leukocyte adhesion after burn injury. In summary, cingulin limits non-perfused area after burn injury and maintains the paracellular barrier of blood vessels. Since edema formation with serious systemic effects is a central problem of burn wounds, understanding the importance of tight junction proteins might help to find new treatment strategies for burn wounds.
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http://dx.doi.org/10.1016/j.mvr.2020.104067DOI Listing
November 2020

Scaffolding proteins of vertebrate apical junctions: structure, functions and biophysics.

Biochim Biophys Acta Biomembr 2020 10 15;1862(10):183399. Epub 2020 Jun 15.

Department of Cell Biology, Faculty of Sciences, University of Geneva, 4 Boulevard d'Yvoy, CH-1211 Geneva, Switzerland. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbamem.2020.183399DOI Listing
October 2020

Cooperative binding of the tandem WW domains of PLEKHA7 to PDZD11 promotes conformation-dependent interaction with tetraspanin 33.

J Biol Chem 2020 07 5;295(28):9299-9312. Epub 2020 May 5.

Department of Cell Biology, Faculty of Sciences, University of Geneva, Geneva, Switzerland

Pleckstrin homology domain-containing A7 (PLEKHA7) is a cytoplasmic protein at adherens junctions that has been implicated in hypertension, glaucoma, and responses to α-toxin. Complex formation between PLEKHA7, PDZ domain-containing 11 (PDZD11), tetraspanin 33, and the α-toxin receptor ADAM metallopeptidase domain 10 (ADAM10) promotes junctional clustering of ADAM10 and α-toxin-mediated pore formation. However, how the N-terminal region of PDZD11 interacts with the N-terminal tandem WW domains of PLEKHA7 and how this interaction promotes tetraspanin 33 binding to the WW1 domain is unclear. Here, we used site-directed mutagenesis, glutathione -transferase pulldown experiments, immunofluorescence, molecular modeling, and docking experiments to characterize the mechanisms driving these interactions. We found that Asp-30 of WW1 and His-75 of WW2 interact through a hydrogen bond and, together with Thr-35 of WW1, form a binding pocket that accommodates a polyproline stretch within the N-terminal PDZD11 region. By strengthening the interactions of the ternary complex, the WW2 domain stabilized the WW1 domain and cooperatively promoted the interaction with PDZD11. Modeling results indicated that, in turn, PDZD11 binding induces a conformational rearrangement, which strengthens the ternary complex, and contributes to enlarging a "hydrophobic hot spot" region on the WW1 domain. The last two lipophilic residues of tetraspanin 33, Trp-283 and Tyr-282, were required for its interaction with PLEKHA7. Docking of the tetraspanin 33 C terminus revealed that it fits into the hydrophobic hot spot region of the accessible surface of WW1. We conclude that communication between the two tandem WW domains of PLEKHA7 and the PLEKHA7-PDZD11 interaction modulate the ligand-binding properties of PLEKHA7.
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http://dx.doi.org/10.1074/jbc.RA120.012987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363125PMC
July 2020

Cell Biology: Tight Junctions as Biomolecular Condensates.

Authors:
Sandra Citi

Curr Biol 2020 01;30(2):R83-R86

Department of Cell Biology, Faculty of Sciences, University of Geneva, 1211-4 Geneva, Switzerland. Electronic address:

Two recent studies report that ZO proteins, the main scaffolding proteins of tight junctions, undergo liquid phase separation. This new concept provides understanding at the mechanistic level of how tight junctions are formed and how they participate in mechanochemical signaling in early development.
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http://dx.doi.org/10.1016/j.cub.2019.11.060DOI Listing
January 2020

The mechanobiology of tight junctions.

Authors:
Sandra Citi

Biophys Rev 2019 Oct 4;11(5):783-793. Epub 2019 Oct 4.

Department of Cell Biology, Faculty of Sciences, University of Geneva, 1211-4, Geneva, Switzerland.

Tight junctions (TJ) play a central role in the homeostasis of epithelial and endothelial tissues, by providing a semipermeable barrier to ions and solutes, by contributing to the maintenance of cell polarity, and by functioning as signaling platforms. TJ are associated with the actomyosin and microtubule cytoskeletons, and the crosstalk with the cytoskeleton is fundamental for junction biogenesis and physiology. TJ are spatially and functionally connected to adherens junctions (AJ), which are essential for the maintenance of tissue integrity. Mechano-sensing and mechano-transduction properties of several AJ proteins have been characterized during the last decade. However, little is known about how mechanical forces act on TJ and their proteins, how TJ control the mechanical properties of cells and tissues, and what are the underlying molecular mechanisms. Here I review recent studies that have advanced our understanding of the relationships between mechanical force and TJ biology.
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http://dx.doi.org/10.1007/s12551-019-00582-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6815314PMC
October 2019

R40.76 binds to the α domain of ZO-1: role of ZO-1 (α+) in epithelial differentiation and mechano-sensing.

Tissue Barriers 2019 22;7(3):e1653748. Epub 2019 Aug 22.

Department of Cell Biology, Faculty of Sciences, University of Geneva , Geneva , Switzerland.

The barrier function of epithelia and endothelia depends on tight junctions, which are formed by the polymerization of claudins on a scaffold of ZO proteins. Two differentially spliced isoforms of ZO-1 have been described, depending on the presence of the α domain, but the function of this domain is unclear. ZO-1 also contains a C-terminal ZU5 domain, which is involved in a mechano-sensitive intramolecular interaction with the central (ZPSG) region of ZO-1. Here we use immunoblotting and immunofluorescence to map the binding sites for commercially available monoclonal and polyclonal antibodies against ZO-1, and for a new polyclonal antibody (R3) that we developed against the ZO-1 C-terminus. We demonstrate that antibody R40.76 binds to the α domain, and the R3 antibody binds to the ZU5 domain. The (α+) isoform of ZO-1 shows higher expression in epithelial versus endothelial cells, and in differentiated versus undifferentiated primary keratinocytes, suggesting a link to epithelial differentiation and a potential molecular adaptation to junctions subjected to stronger mechanical forces. These results provide new tools and hypotheses to investigate the role of the α and ZU5 domains in ZO-1 mechano-sensing and dynamic interactions with the cytoskeleton and junctional ligands.
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http://dx.doi.org/10.1080/21688370.2019.1653748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748370PMC
July 2020

LncRNA EPR controls epithelial proliferation by coordinating Cdkn1a transcription and mRNA decay response to TGF-β.

Nat Commun 2019 04 29;10(1):1969. Epub 2019 Apr 29.

Gene Expression Regulation Laboratory, IRCCS Ospedale Policlinico San Martino, 16132, Genova, Italy.

Long noncoding RNAs (lncRNAs) are emerging as regulators of fundamental biological processes. Here we report on the characterization of an intergenic lncRNA expressed in epithelial tissues which we termed EPR (Epithelial cell Program Regulator). EPR is rapidly downregulated by TGF-β and its sustained expression largely reshapes the transcriptome, favors the acquisition of epithelial traits, and reduces cell proliferation in cultured mammary gland cells as well as in an animal model of orthotopic transplantation. EPR generates a small peptide that localizes at epithelial cell junctions but the RNA molecule per se accounts for the vast majority of EPR-induced gene expression changes. Mechanistically, EPR interacts with chromatin and regulates Cdkn1a gene expression by affecting both its transcription and mRNA decay through its association with SMAD3 and the mRNA decay-promoting factor KHSRP, respectively. We propose that EPR enables epithelial cells to control proliferation by modulating waves of gene expression in response to TGF-β.
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http://dx.doi.org/10.1038/s41467-019-09754-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488594PMC
April 2019

A Dock-and-Lock Mechanism Clusters ADAM10 at Cell-Cell Junctions to Promote α-Toxin Cytotoxicity.

Cell Rep 2018 11;25(8):2132-2147.e7

Department of Cell Biology, Faculty of Sciences, University of Geneva, 1211-4 Geneva, Switzerland; Institute for Genetics and Genomics of Geneva (iGE3), University of Geneva, 1211-4 Geneva, Switzerland. Electronic address:

We previously identified PLEKHA7 and other junctional proteins as host factors mediating death by S. aureus α-toxin, but the mechanism through which junctions promote toxicity was unclear. Using cell biological and biochemical methods, we now show that ADAM10 is docked to junctions by its transmembrane partner Tspan33, whose cytoplasmic C terminus binds to the WW domain of PLEKHA7 in the presence of PDZD11. ADAM10 is locked at junctions through binding of its cytoplasmic C terminus to afadin. Junctionally clustered ADAM10 supports the efficient formation of stable toxin pores. Instead, disruption of the PLEKHA7-PDZD11 complex inhibits ADAM10 and toxin junctional clustering. This promotes toxin pore removal from the cell surface through an actin- and macropinocytosis-dependent process, resulting in cell recovery from initial injury and survival. These results uncover a dock-and-lock molecular mechanism to target ADAM10 to junctions and provide a paradigm for how junctions regulate transmembrane receptors through their clustering.
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http://dx.doi.org/10.1016/j.celrep.2018.10.088DOI Listing
November 2018

The role of microtubules in the regulation of epithelial junctions.

Tissue Barriers 2018 5;6(3):1539596. Epub 2018 Nov 5.

a Department of Cell Biology, Faculty of Sciences and Institute for Genetics and Genomics in Geneva (iGE3) , University of Geneva , Geneva , Switzerland.

The cytoskeleton is crucially important for the assembly of cell-cell junctions and the homeostatic regulation of their functions. Junctional proteins act, in turn, as anchors for cytoskeletal filaments, and as regulators of cytoskeletal dynamics and signalling proteins. The cross-talk between junctions and the cytoskeleton is critical for the morphogenesis and physiology of epithelial and other tissues, but is not completely understood. Microtubules are implicated in the delivery of junctional proteins to cell-cell contact sites, in the differentiation and spatial organization of the cytoplasm, and in the stabilization of the barrier and adhesive functions of junctions. Here we focus on the relationships between microtubules and junctions of vertebrate epithelial cells. We highlight recent discoveries on the molecular underpinnings of microtubule-junction interactions, and report new data about the interaction of cingulin and paracingulin with microtubules. We also propose a possible new role of junctions as "molecular sinks" for microtubule-associated signalling proteins.
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http://dx.doi.org/10.1080/21688370.2018.1539596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389124PMC
February 2019

Intestinal barriers protect against disease.

Authors:
Sandra Citi

Science 2018 03;359(6380):1097-1098

Department of Cell Biology, Faculty of Sciences, and Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, Switzerland 30, Quai Ernest Ansermet, 1205 Geneva, Switzerland.

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http://dx.doi.org/10.1126/science.aat0835DOI Listing
March 2018

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

Curr Biol 2017 Dec 5;27(24):3783-3795.e8. Epub 2017 Dec 5.

Department of Cell Biology, Faculty of Sciences, University of Geneva, 30 Quai Ernest Ansermet, 1211 Geneva 4, Switzerland; Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, 30 Quai Ernest Ansermet, 1211 Geneva 4, Switzerland. Electronic address:

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. Pull-down experiments show that interactions between N-terminal (ZPSG) and C-terminal domains of ZO-1 prevent binding of DbpA to the ZPSG, suggesting that force-dependent intra-molecular interactions regulate ZPSG binding to ligands within cells. In vivo and in vitro experiments also suggest that ZO-1 heterodimerization with ZO-2 promotes the stretched conformation and ZPSG interaction with ligands. Magnetic tweezers single-molecule experiments suggest that pN-scale tensions (∼2-4 pN) are sufficient to maintain the stretched conformation of ZO-1, while keeping its structured domains intact, and that 5-20 pN force is required to disrupt the interaction between the extreme C-terminal and the ZPSG domains of ZO-1. We propose that tensile forces regulate epithelial homeostasis by activating ZO proteins through stretching, to control the junctional recruitment and downstream signaling of their interactors.
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http://dx.doi.org/10.1016/j.cub.2017.11.014DOI Listing
December 2017

Cell-specific diversity in the expression and organization of cytoplasmic plaque proteins of apical junctions.

Ann N Y Acad Sci 2017 10 15;1405(1):160-176. Epub 2017 Jun 15.

Department of Cell Biology, Faculty of Sciences, Institute of Genetics and Genomics in Geneva (iGE3), Geneva, Switzerland.

Tight and adherens junctions play critical roles in the barrier, adhesion, and signaling functions of epithelial and endothelial cells. How the molecular organization of these junctions is tuned to the widely diverse physiological requirements of each tissue type is not well understood. Here, we address this question by examining the expression, localization, and interactions of major cytoplasmic plaque proteins of tight and adherens junctions in different cultured epithelial and endothelial cell lines. Immunoblotting and immunofluorescence analyses show that the expression profiles of cingulin, paracingulin, ZO-1, ZO-2, ZO-3, PLEKHA7, afadin, PDZD11, p120-catenin, and α-catenin, as well as the transmembrane junctional proteins occludin, E-cadherin, and VE-cadherin, are significantly diverse when comparing kidney cells (MDCK, mCCD), keratinocytes (HaCaT), lung carcinoma (A427, A549), and endothelium-derived cells (bEnd.3, meEC, H5V). Proximity ligation and co-immunoprecipitation assays show that PLEKHA7 and PDZD11 are significantly more associated with the tight junction proteins cingulin and ZO-1 in aortic endothelium-derived (meEC) cells but not kidney collecting duct epithelial (mCCD) cells. These results provide evidence that the cytoplasmic plaques of tight and adherens junctions are diverse in their composition and molecular architecture and establish a conceptual framework by which we can rationally address the mechanisms of tissue-dependent junction physiology and signaling by cytoplasmic junctional proteins.
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http://dx.doi.org/10.1111/nyas.13391DOI Listing
October 2017

The role of apical cell-cell junctions and associated cytoskeleton in mechanotransduction.

Biol Cell 2017 Apr 13;109(4):139-161. Epub 2017 Mar 13.

Department of Cell Biology, Institute of Genomics and Genetics of Geneva (iGE3), University of Geneva, Geneva, Switzerland.

Tissues of multicellular organisms are characterised by several types of specialised cell-cell junctions. In vertebrate epithelia and endothelia, tight and adherens junctions (AJ) play critical roles in barrier and adhesion functions, and are connected to the actin and microtubule cytoskeletons. The interaction between junctions and the cytoskeleton is crucial for tissue development and physiology, and is involved in the molecular mechanisms governing cell shape, motility, growth and signalling. The machineries which functionally connect tight and AJ to the cytoskeleton comprise proteins which either bind directly to cytoskeletal filaments, or function as adaptors for regulators of the assembly and function of the cytoskeleton. In the last two decades, specific cytoskeleton-associated junctional molecules have been implicated in mechanotransduction, revealing the existence of multimolecular complexes that can sense mechanical cues and translate them into adaptation to tensile forces and biochemical signals. Here, we summarise the current knowledge about the machineries that link tight and AJ to actin filaments and microtubules, and the molecular basis for mechanotransduction at epithelial and endothelial AJ.
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http://dx.doi.org/10.1111/boc.201600075DOI Listing
April 2017

Grete Kellenberger-Gujer: Molecular biology research pioneer.

Bacteriophage 2016 Apr-Jun;6(2):e1148805. Epub 2016 Apr 5.

Department of Medicine, Division of Infectious Disease, University of California , San Diego, La Jolla, CA.

Grete Kellenberger-Gujer was a Swiss molecular biologist who pioneered fundamental studies of bacteriophage in the mid-20(th) century at the University of Geneva. Her life and career stories are reviewed here, focusing on her fundamental contributions to our early understanding of phage biology via her insightful analyses of phenomena such as the lysogenic state of a temperate phage (λ), genetic recombination, radiation's in vivo consequences, and DNA restriction-modification; on her creative personality and interactions with peers; and how her academic advancement was affected by gender, societal conditions and cultural attitudes of the time. Her story is important scientifically, putting into perspective features of the scientific community from just before the molecular biology era started through its early years, and also sociologically, in illustrating the numerous "glass ceilings" that, especially then, often hampered the advancement of creative women.
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http://dx.doi.org/10.1080/21597081.2016.1173168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4951002PMC
September 2016

Role of Cingulin in Agonist-induced Vascular Endothelial Permeability.

J Biol Chem 2016 Nov 2;291(45):23681-23692. Epub 2016 Sep 2.

From the Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, Illinois 60637 and

Agonist-induced activation of Rho GTPase signaling leads to endothelial cell (EC) permeability and may culminate in pulmonary edema, a devastating complication of acute lung injury. Cingulin is an adaptor protein first discovered in epithelium and is involved in the organization of the tight junctions. This study investigated the role of cingulin in control of agonist-induced lung EC permeability via interaction with RhoA-specific activator GEF-H1. The siRNA-induced cingulin knockdown augmented thrombin-induced EC permeability monitored by measurements of transendothelial electrical resistance and endothelial cell permeability for macromolecules. Increased thrombin-induced permeability in ECs with depleted cingulin was associated with increased activation of GEF-H1 and RhoA detected in pulldown activation assays. Increased GEF-H1 association with cingulin was essential for down-regulation of thrombin-induced RhoA barrier disruptive signaling. Using cingulin-truncated mutants, we determined that GEF-H1 interaction with the rod + tail domain of cingulin was required for inactivation of GEF-H1 and endothelial cell barrier preservation. The results demonstrate the role for association of GEF-H1 with cingulin as the mechanism of RhoA pathway inactivation and rescue of EC barrier after agonist challenge.
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http://dx.doi.org/10.1074/jbc.M116.720763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095421PMC
November 2016

Cingulin and actin mediate midbody-dependent apical lumen formation during polarization of epithelial cells.

Nat Commun 2016 08 3;7:12426. Epub 2016 Aug 3.

Department of Cell and Developmental Biology, School of Medicine, Anschutz Medical Campus, University of Colorado Denver, Aurora, Colorado 80045, USA.

Coordinated polarization of epithelial cells is a key step during morphogenesis that leads to the formation of an apical lumen. Rab11 and its interacting protein FIP5 are necessary for the targeting of apical endosomes to the midbody and apical membrane initiation site (AMIS) during lumenogenesis. However, the machinery that mediates AMIS establishment and FIP5-endosome targeting remains unknown. Here we identify a FIP5-interacting protein, Cingulin, which localizes to the AMIS and functions as a tether mediating FIP5-endosome targeting. We analysed the machinery mediating AMIS recruitment to the midbody and determined that both branched actin and microtubules are required for establishing the site of the nascent lumen. We demonstrate that the Rac1-WAVE/Scar complex mediates Cingulin recruitment to the AMIS by inducing branched actin formation, and that Cingulin directly binds to microtubule C-terminal tails through electrostatic interactions. We propose a new mechanism for apical endosome targeting and AMIS formation around the midbody during epithelial lumenogenesis.
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http://dx.doi.org/10.1038/ncomms12426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4976216PMC
August 2016

PLEKHA7: Cytoskeletal adaptor protein at center stage in junctional organization and signaling.

Int J Biochem Cell Biol 2016 06 9;75:112-6. Epub 2016 Apr 9.

Department of Cell Biology, University of Geneva, Geneva, Switzerland; The Institute for Genetics and Genomics in Geneva (iGE3), University of Geneva, Geneva, Switzerland. Electronic address:

PLEKHA7 is a recently characterized component of the cytoplasmic region of epithelial adherens junctions (AJ). It comprises two WW domains, a pleckstrin-homology domain, and proline-rich and coiled-coil domains. PLEKHA7 interacts with cytoplasmic components of the AJ (p120-catenin, paracingulin, afadin), stabilizes the E-cadherin complex by linking it to the minus ends of noncentrosomal microtubules, and stabilizes junctional nectins through the newly identified interactor PDZD11. Similarly to afadin, and unlike E-cadherin and p120-catenin, the localization of PLEKHA7 at AJ is strictly zonular (in the zonula adhaerens subdomain of AJ), and does not extend along the basolateral contacts. Genome-wide association studies and experiments on animal and cellular models show that although PLEKHA7 is not required for organism viability, it is implicated in cardiovascular physiology, hypertension, primary angle closure glaucoma, susceptibility to staphylococcal α-toxin, and epithelial morphogenesis and growth. Thus, PLEKHA7 is a cytoskeletal adaptor protein important for AJ organization, and at the center of junction-associated signaling pathways which fine-tune important pathophysiological processes.
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http://dx.doi.org/10.1016/j.biocel.2016.04.001DOI Listing
June 2016

PLEKHA7 Recruits PDZD11 to Adherens Junctions to Stabilize Nectins.

J Biol Chem 2016 May 4;291(21):11016-29. Epub 2016 Apr 4.

From the Department of Cell Biology and Institute for Genetics and Genomics in Geneva (iGE3), University of Geneva, 1211-4 Geneva, Switzerland,

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. We show that PDZD11 (17 kDa) is expressed in epithelial and endothelial cells, where it forms a complex with PLEKHA7, as determined by co-immunoprecipitation analysis. The N-terminal Trp-Trp (WW) domain of PLEKHA7 interacts directly with the N-terminal 44 amino acids of PDZD11, as shown by GST-pulldown assays. Immunofluorescence analysis shows that PDZD11 is localized at adherens junctions in a PLEKHA7-dependent manner, because its junctional localization is abolished by knock-out of PLEKHA7, and is rescued by re-expression of exogenous PLEKHA7. The junctional recruitment of nectin-1 and nectin-3 and their protein levels are decreased via proteasome-mediated degradation in epithelial cells where either PDZD11 or PLEKHA7 have been knocked-out. PDZD11 forms a complex with nectin-1 and nectin-3, and its PDZ domain interacts directly with the PDZ-binding motif of nectin-1. PDZD11 is required for the efficient assembly of apical junctions of epithelial cells at early time points in the calcium-switch model. These results show that the PLEKHA7-PDZD11 complex stabilizes nectins to promote efficient early junction assembly and uncover a new molecular mechanism through which PLEKHA7 recruits PDZ-binding membrane proteins to epithelial adherens junctions.
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http://dx.doi.org/10.1074/jbc.M115.712935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4900252PMC
May 2016

Evidence That Cingulin Regulates Endothelial Barrier Function In Vitro and In Vivo.

Arterioscler Thromb Vasc Biol 2016 Apr 28;36(4):647-54. Epub 2016 Jan 28.

From the Skin and Endothelium Research Division (SERD), Department of Dermatology (K.S., S.R., M.G., H.P.F., R.F., P.P.), Core Facility Imaging (S.R., M.G.), Department of Cardiac Surgery (A.H.), Department of Medicine I, Institute of Cancer Research, Comprehensive Cancer Center (M.S.), Department of Surgery (C.B.), and Department of Dermatology (D.F.), Medical University of Vienna, Vienna, Austria; and Department of Cell Biology and Institute of Genetics and Genomics in Geneva, University of Geneva, Switzerland (S.C.).

Objective: Cingulin is a cytoplasmic component of tight junctions. Although modulation of cingulin levels in cultured epithelial model systems has no significant effect on barrier function, evidence from cingulin knockout mice suggests that cingulin may be involved in the regulation of the behavior of epithelial or endothelial cells. Here, we investigate the role of cingulin in the barrier function of endothelial cells.

Approach And Results: We show that cingulin is expressed in human endothelial cells of the skin, brain, and lung in vivo and in vitro. Endothelial cingulin colocalizes and coimmunoprecipitates with the tight junction proteins zonula occludens-1 and guanine nucleotide exchange factor-H1. Cingulin overexpression in human umbilical vein endothelial cell induces tight junction formation, increases transendothelial electric resistance, and strengthens barrier function for low and high molecular weight tracers. In contrast, cultured endothelial cells lacking cingulin are more permeable for low molecular weight tracers. In cingulin knockout mice, neurons of the area postrema and Purkinje cells show an increased uptake of small molecular weight tracers indicating decreased barrier function at these sites.

Conclusions: We demonstrate that cingulin participates in the modulation of endothelial barrier function both in human cultured cells in vitro and in mouse brains in vivo. Understanding the role of cingulin in maintaining tight barriers in endothelia may allow developing new strategies for the treatment of vascular leak syndromes.
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http://dx.doi.org/10.1161/ATVBAHA.115.307032DOI Listing
April 2016

The adherens junctions control susceptibility to Staphylococcus aureus α-toxin.

Proc Natl Acad Sci U S A 2015 Nov 21;112(46):14337-42. Epub 2015 Oct 21.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305; Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305

Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin. We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7(-/-) mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections.
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http://dx.doi.org/10.1073/pnas.1510265112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655540PMC
November 2015

Distinct E-cadherin-based complexes regulate cell behaviour through miRNA processing or Src and p120 catenin activity.

Nat Cell Biol 2015 Sep 24;17(9):1145-57. Epub 2015 Aug 24.

Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Mayo Clinic, 4500 San Pablo Road, Jacksonville, Florida 32224, USA.

E-cadherin and p120 catenin (p120) are essential for epithelial homeostasis, but can also exert pro-tumorigenic activities. Here, we resolve this apparent paradox by identifying two spatially and functionally distinct junctional complexes in non-transformed polarized epithelial cells: one growth suppressing at the apical zonula adherens (ZA), defined by the p120 partner PLEKHA7 and a non-nuclear subset of the core microprocessor components DROSHA and DGCR8, and one growth promoting at basolateral areas of cell-cell contact containing tyrosine-phosphorylated p120 and active Src. Recruitment of DROSHA and DGCR8 to the ZA is PLEKHA7 dependent. The PLEKHA7-microprocessor complex co-precipitates with primary microRNAs (pri-miRNAs) and possesses pri-miRNA processing activity. PLEKHA7 regulates the levels of select miRNAs, in particular processing of miR-30b, to suppress expression of cell transforming markers promoted by the basolateral complex, including SNAI1, MYC and CCND1. Our work identifies a mechanism through which adhesion complexes regulate cellular behaviour and reveals their surprising association with the microprocessor.
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http://dx.doi.org/10.1038/ncb3227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4975377PMC
September 2015

The Expression of the Zonula Adhaerens Protein PLEKHA7 Is Strongly Decreased in High Grade Ductal and Lobular Breast Carcinomas.

PLoS One 2015 13;10(8):e0135442. Epub 2015 Aug 13.

Department of Cell Biology, University of Geneva, Geneva, Switzerland; Institute of Genomics and Genetics of Geneva (iGE3), University of Geneva, Geneva, Switzerland.

PLEKHA7 is a junctional protein, which participates in a complex that stabilizes E-cadherin at the zonula adhaerens. Since E-cadherin is involved in epithelial morphogenesis, signaling, and tumor progression, we explored PLEKHA7 expression in cancer. PLEKHA7 expression was assessed in invasive ductal and lobular carcinomas of the breast by immunohistochemistry, immunofluorescence and quantitative RT-PCR. PLEKHA7 was detected at epithelial junctions of normal mammary ducts and lobules, and of tubular and micropapillary structures within G1 and G2 ductal carcinomas. At these junctions, the localization of PLEKHA7 was along the circumferential belt (zonula adhaerens), and only partially overlapping with that of E-cadherin, p120ctn and ZO-1, as shown previously in rodent tissues. PLEKHA7 immunolabeling was strongly decreased in G3 ductal carcinomas and undetectable in lobular carcinomas. PLEKHA7 mRNA was detected in both ductal and lobular carcinomas, with no observed correlation between mRNA levels and tumor type or grade. In summary, PLEKHA7 is a junctional marker of epithelial cells within tubular structures both in normal breast tissue and ductal carcinomas, and since PLEKHA7 protein but not mRNA expression is strongly decreased or lost in high grade ductal carcinomas and in lobular carcinomas, loss of PLEKHA7 is a newly characterized feature of these carcinomas.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0135442PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4535953PMC
May 2016

Epithelial junctions and Rho family GTPases: the zonular signalosome.

Small GTPases 2014 ;5(4):1-15

a Department of Cell Biology ; University of Geneva ; Geneva , Switzerland.

The establishment and maintenance of epithelial cell-cell junctions is crucially important to regulate adhesion, apico-basal polarity and motility of epithelial cells, and ultimately controls the architecture and physiology of epithelial organs. Junctions are supported, shaped and regulated by cytoskeletal filaments, whose dynamic organization and contractility are finely tuned by GTPases of the Rho family, primarily RhoA, Rac1 and Cdc42. Recent research has identified new molecular mechanisms underlying the cross-talk between these GTPases and epithelial junctions. Here we briefly summarize the current knowledge about the organization, molecular evolution and cytoskeletal anchoring of cell-cell junctions, and we comment on the most recent advances in the characterization of the interactions between Rho GTPases and junctional proteins, and their consequences with regards to junction assembly and regulation of cell behavior in vertebrate model systems. The concept of "zonular signalosome" is proposed, which highlights the close functional relationship between proteins of zonular junctions (zonulae occludentes and adhaerentes) and the control of cytoskeletal organization and signaling through Rho GTPases, transcription factors, and their effectors.
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http://dx.doi.org/10.4161/21541248.2014.973760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4601189PMC
August 2015

Toll-like receptor 2 regulates the barrier function of human bronchial epithelial monolayers through atypical protein kinase C zeta, and an increase in expression of claudin-1.

Tissue Barriers 2014 12;2:e29166. Epub 2014 May 12.

School of Pharmaceutical Sciences; University of Geneva; University of Lausanne; Geneva, Switzerland.

We investigated the role of Toll-like receptor (TLR) 2 in maintaining the integrity of the airway epithelial barrier using the human bronchial epithelial cell line Calu-3. Activation of TLR2 by its ligands, Pam3CysSK4 and Peptidoglycan showed a concentration dependent increase in epithelial barrier function, as measured by transepithelial electrical resistance (TEER). This was confirmed by a decrease in paracellular flux of fluorescein sodium. This TLR2 induced increase in TEER was significantly reduced by pretreatment with polyclonal anti-human TLR2-neutralizing antibody. TLR2 stimulation in Calu-3 cell monolayers resulted in an increased expression of the tight junction proteins claudin-1 and ZO-1, and a decreased expression of occludin, at both the mRNA and protein levels. A pseudosubstrate inhibitor to PKCζ significantly prevented the TLR2 mediated increase in barrier function. It also prevented the increase in claudin-1 in a concentration dependent manner up to 1 µM. TLR2 stimulation led to an increase in phosphorylation of atypical PKC ζ, which was prevented by the pseudosubstrate inhibitor in a concentration dependent manner. Taken together, our observations support a model whereby increased tight junction barrier function induced by activation of TLR2 occurs through increased expression of claudin-1, and through modulation of PKC ζ activity.
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http://dx.doi.org/10.4161/tisb.29166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4117686PMC
August 2014

ZO proteins redundantly regulate the transcription factor DbpA/ZONAB.

J Biol Chem 2014 Aug 1;289(32):22500-11. Epub 2014 Jul 1.

From the Department of Cell Biology, University of Geneva, 1211 Geneva, Switzerland,

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. To address this discrepancy, we examined the localization and activity of DbpA and YAP in Madin-Darby canine kidney cells depleted either of ZO-1, or one of the related proteins ZO-2 and ZO-3 (ZO proteins), or all three together. Depletion of only one ZO protein had no effect on DbpA localization and activity, whereas depletion of ZO-1 and ZO-2, which is associated with reduced ZO-3 expression, resulted in increased DbpA localization in the cytoplasm. Only depletion of ZO-2 reduced the nuclear import of YAP. Mammary epithelial (Eph4) cells KO for ZO-1 showed junctional DbpA, demonstrating that ZO-1 is not required to sequester DbpA at junctions. However, further depletion of ZO-2 in Eph4 ZO-1KO cells, which do not express ZO-3, caused decreased junctional localization and expression of DbpA, which were rescued by the proteasome inhibitor MG132. In vitro binding assays showed that full-length ZO-1 does not interact with DbpA. These results show that ZO-2 is implicated in regulating the nuclear shuttling of YAP, whereas ZO proteins redundantly control the junctional retention and stability of DbpA, without affecting its shuttling to the nucleus.
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http://dx.doi.org/10.1074/jbc.M114.556449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139256PMC
August 2014

PLEKHA7 modulates epithelial tight junction barrier function.

Tissue Barriers 2014 Jan 2;2(1):e28755. Epub 2014 Apr 2.

Departments of Cell Biology and Molecular Biology; University of Geneva; Geneva ; Switzerland Institute of Genetics and Genomics of Geneva; University of Geneva; Geneva, Switzerland.

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. All constructs were localized at junctions, but constructs lacking the C-terminal region were also distributed diffusely in the cytoplasm. Inducible expression of PLEKHA7 constructs did not affect the expression and localization of TJ proteins, the steady-state value of transepithelial resistance (TER), the development of TER during the calcium switch, and the flux of large molecules across confluent monolayers. In contrast, expression of three out of four constructs resulted both in enhanced recruitment of E-cadherin and associated proteins at the apical ZA and at lateral puncta adherentia (PA), a decreased TER at 18 h after assembly at normal calcium, and an attenuation in the fall in TER after extracellular calcium removal. This latter effect was inhibited when cells were treated with nocodazole. Immunoprecipitation analysis showed that PLEKHA7 forms a complex with the cytoplasmic TJ proteins ZO-1 and cingulin, and this association does not depend on the integrity of microtubules. These results suggest that PLEKHA7 modulates the dynamics of assembly and disassembly of the TJ barrier, through E-cadherin protein complex- and microtubule-dependent mechanisms.
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http://dx.doi.org/10.4161/tisb.28755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4022608PMC
January 2014

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

Mol Biol Cell 2014 Jul 7;25(13):1995-2005. Epub 2014 May 7.

Department of Molecular Biology, University of Geneva, CH-1211 Geneva, SwitzerlandDepartment of Cell Biology, University of Geneva, CH-1211 Geneva, SwitzerlandInstitute of Genetics and Genomics in Geneva, University of Geneva, CH-1211 Geneva, Switzerland

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. Here we show that, unexpectedly, Madin-Darby canine kidney epithelial cells depleted of both CGN and CGNL1 (double-KD cells) display normal Rac1 activation and tight junction (TJ) formation, despite decreased junctional recruitment of the Rac1 activator Tiam1. The expression of the Rac1 inhibitor MgcRacGAP is decreased in double-KD cells, and the barrier development and Rac1 activation phenotypes are rescued by exogenous expression of MgcRacGAP. MgcRacGAP colocalizes with CGN and CGNL1 at TJs and forms a complex and interacts directly in vitro with CGN and CGNL1. Depletion of either CGN or CGNL1 in epithelial cells results in decreased junctional localization of MgcRacGAP but not of ECT2, a centralspindlin-interacting Rho GEF. These results provide new insight into coordination of Rho-family GTPase activities at junctions, since apical accumulation of CGN and CGNL1 at TJs during junction maturation provides a mechanism to spatially restrict down-regulation of Rac1 activation through the recruitment of MgcRacGAP.
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http://dx.doi.org/10.1091/mbc.E13-11-0680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072573PMC
July 2014

The junctional proteins cingulin and paracingulin modulate the expression of tight junction protein genes through GATA-4.

PLoS One 2013 7;8(2):e55873. Epub 2013 Feb 7.

Department of Molecular Biology, University of Geneva, Geneva, Switzerland.

The cytoplamic junctional proteins cingulin and paracingulin have been implicated in the regulation of gene expression in different cultured cell models. In renal epithelial MDCK cells, depletion of either protein results in a Rho-dependent increase in the expression of claudin-2. Here we examined MDCK cell clones depleted of both cingulin and paracingulin (double-KD cells), and we found that unexpectedly the expression of claudin-2, and also the expression of ZO-3 and claudin-3, were decreased, while RhoA activity was still higher than in control cells. The decreased expression of claudin-2 and other TJ proteins in double-KD cells correlated with reduced levels of the transcription factor GATA-4, and was rescued by overexpression of GATA-4, but not by inhibiting RhoA activity. These results indicate that in MDCK cells GATA-4 is required for the expression of claudin-2 and other TJ proteins, and that maintenance of GATA-4 expression requires either cingulin or paracingulin. These results and previous studies suggest a model whereby cingulin and paracingulin redundantly control the expression of specific TJ proteins through distinct GATA-4- and RhoA-dependent mechanisms, and that in the absence of sufficient levels of GATA-4 the RhoA-mediated upregulation of claudin-2 is inhibited.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0055873PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567034PMC
August 2013

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.

J Cell Sci 2012 Nov 3;125(Pt 21):5005-14. Epub 2012 Sep 3.

Department of Molecular Biology, University of Geneva, CH-1211 Geneva, Switzerland.

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. Immunohistochemistry, immunofluorescence, electron microscopy and permeability assays of epithelial tissues of CGN(-/-) mice show no cingulin labeling at junctions, a normal localization of TJ proteins, and normal TJ structure and barrier function. Microarray analysis of intestinal cells does not show significant changes in gene expression between CGN(-/-) and CGN(+/+) mice, whereas immunoblotting analysis shows a twofold increase in the levels of claudin-2 protein in the duodenum and the kidney of CGN(-/-) mice, compared to CGN(+/+) littermates. Furthermore, CGN(-/-) mice show an exacerbated response to the ulcerogenic action of cysteamine, whereas acute injury of the colon by dextran sodium sulfate elicits undistinguishable responses in CGN(-/-) and CGN(+/+) mice. We conclude that at the organism level cingulin is dispensable for the structure and barrier function of TJ, and is embedded in signaling networks that control the expression of claudin-2, and the mucosal response to acute injury in the duodenum.
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http://dx.doi.org/10.1242/jcs.101261DOI Listing
November 2012

The control of gene expression and cell proliferation by the epithelial apical junctional complex.

Essays Biochem 2012 ;53:83-93

†Department of Molecular Biology, University of Geneva, 4 Boulevard d'Yvoy, CH-1211-4 Geneva, Switzerland.

The AJC (apical junctional complex) of vertebrate epithelial cells orchestrates cell-cell adhesion and tissue barrier function. In addition, it plays a pivotal role in signalling. Several protein components of the AJC, e.g. the cytoplasmic proteins β-catenin, p120-catenin and ZO (Zonula Occludens)-2, can shuttle to the nucleus, where they interact with transcription factors to regulate gene expression and cell proliferation. Other junctional proteins, e.g. angiomotin, α-catenin and cingulin, are believed to act by sequestering either transcription factors, such as YAP (Yes-associated protein), or regulators of small GTPases, such as GEF (guanine-nucleotide-exchange factor)-H1, at junctions. The signalling activities of AJC proteins are triggered by different extracellular and intracellular cues, including cell density, and physiological or pathological activation of developmentally regulated pathways, such as the Wnt pathway. The interplay between junctional protein complexes, the actin cytoskeleton and signalling pathways is of crucial importance in the regulation of gene expression and cell proliferation.
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http://dx.doi.org/10.1042/bse0530083DOI Listing
December 2012