scholarly journals Characterization of Porphyromonas gingivalis-Induced Degradation of Epithelial Cell Junctional Complexes

2000 ◽  
Vol 68 (3) ◽  
pp. 1441-1449 ◽  
Author(s):  
Jannet Katz ◽  
Vijaya Sambandam ◽  
John H. Wu ◽  
Suzanne M. Michalek ◽  
Daniel F. Balkovetz
Keyword(s):  
Epithelial Cell ◽  
Mdck Cell ◽  
Mdck Cells ◽  
Transmembrane Proteins ◽  
Cell Junctions ◽  
Epithelial Barrier ◽  
Β1 Integrin ◽  
E Cadherin ◽  
Cell Cell

ABSTRACT Porphyromonas gingivalis is considered among the etiological agents of human adult periodontitis. Although in vitro studies have shown that P. gingivalis has the ability to invade epithelial cell lines, its effect on the epithelial barrier junctions is not known. Immunofluorescence analysis of human gingival epithelial cells confirmed the presence of tight-junction (occludin), adherens junction (E-cadherin), and cell-extracellular matrix junction (β1-integrin) transmembrane proteins. These transmembrane proteins are expressed in Madin-Darby canine kidney (MDCK) cells. In addition, MDCK cells polarize and therefore serve as a useful in vitro model for studies on the epithelial cell barrier. Using the MDCK cell system, we examined the effect of P. gingivalis on epithelial barrier function. Exposure of the basolateral surfaces of MDCK cells to P. gingivalis (>109 bacteria/ml) resulted in a decrease in transepithelial resistance. Immunofluorescence microscopy demonstrated decreases in the amounts of immunoreactive occludin, E-cadherin, and β1-integrin at specific times which were related to a disruption of cell-cell junctions in MDCK cells exposed to basolateralP. gingivalis. Disruption of cell-cell junctions was also observed upon apical exposure to bacteria; however, the effects took longer than those seen upon basolateral exposure. Cell viability was not affected by either basolateral or apical exposure to P. gingivalis. Western blot analysis demonstrated hydrolysis of occludin, E-cadherin, and β1-integrin in lysates derived from MDCK cells exposed to P. gingivalis. Immunoprecipitated occludin and E-cadherin molecules from MDCK cell lysates were also degraded by P. gingivalis, suggesting a bacterial protease(s) capable of cleaving these epithelial junction transmembrane proteins. Collectively, these data suggest thatP. gingivalis is able to invade the deeper structures of connective tissues via a paracellular pathway by degrading epithelial cell-cell junction complexes, thus allowing the spread of the bacterium. These results also indicate the importance of a critical threshold concentration of P. gingivalis to initiate epithelial barrier destruction.

scholarly journals Remodeling the cell surface distribution of membrane proteins during the development of epithelial cell polarity.

1992 ◽  
Vol 116 (4) ◽  
pp. 889-899 ◽  
Author(s):  
D A Wollner ◽  
K A Krzeminski ◽  
W J Nelson
Keyword(s):  
Epithelial Cell ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Cell ◽  
Cell Contact ◽  
Surface Polarity ◽  
Lateral Membrane ◽  
E Cadherin ◽  
Cell Cell

The development of polarized epithelial cells from unpolarized precursor cells follows induction of cell-cell contacts and requires resorting of proteins into different membrane domains. We show that in MDCK cells the distributions of two membrane proteins, Dg-1 and E-cadherin, become restricted to the basal-lateral membrane domain within 8 h of cell-cell contact. During this time, however, 60-80% of newly synthesized Dg-1 and E-cadherin is delivered directly to the forming apical membrane and then rapidly removed, while the remainder is delivered to the basal-lateral membrane and has a longer residence time. Direct delivery of greater than 95% of these proteins from the Golgi complex to the basal-lateral membrane occurs greater than 48 h later. In contrast, we show that two apical proteins are efficiently delivered and restricted to the apical cell surface within 2 h after cell-cell contact. These results provide insight into mechanisms involved in the development of epithelial cell surface polarity, and the establishment of protein sorting pathways in polarized cells.

scholarly journals Hydrolysis of Epithelial Junctional Proteins by Porphyromonas gingivalis Gingipains

2002 ◽  
Vol 70 (5) ◽  
pp. 2512-2518 ◽  
Author(s):  
Jannet Katz ◽  
Qiu-Bo Yang ◽  
Ping Zhang ◽  
Jan Potempa ◽  
James Travis ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Porphyromonas Gingivalis ◽  
Catalytic Domain ◽  
Mdck Cells ◽  
Adherens Junction ◽  
Cell Junction ◽  
Critical Threshold ◽  
Hydrolysis Of ◽  
E Cadherin ◽  
Cell Cell

ABSTRACT Porphyromonas gingivalis has been implicated as an etiologic agent of adult periodontitis. We have previously shown that P. gingivalis can degrade the epithelial cell-cell junction complexes, thus suggesting that this bacterium can invade the underlying connective tissues via a paracellular pathway. However, the precise mechanism(s) involved in this process has not been elucidated. The purpose of this study was to determine if the arginine- and lysine-specific gingipains of P. gingivalis (i.e., HRgpA and RgpB, and Kgp, respectively) were responsible for the degradation of E-cadherin, the cell-cell adhesion protein in the adherens junctions. In addition, we compared the degradative abilities of the whole gingipains HRgpA and Kgp to those of their catalytic domains alone. In these studies, immunoprecipitated E-cadherin as well as monolayers of polarized Madin-Darby canine kidney (MDCK) epithelial cell cultures were incubated with the gingipains and hydrolysis of E-cadherin was assessed by Western blot analysis. Incubation of P. gingivalis cells with immunoprecipitated E-cadherin resulted in degradation, whereas prior exposure of P. gingivalis cells to leupeptin and especially acetyl-Leu-Val-Lys-aldehyde (which are arginine- and lysine-specific inhibitors, respectively) reduced this activity. Furthermore, incubation of E-cadherin immunoprecipitates with the different gingipains resulted in an effective and similar hydrolysis of the protein. However, when monolayers of MDCK cells were exposed to the gingipains, Kgp was most effective in hydrolyzing the E-cadherin molecules in the adherens junction. Kgp was more effective than its catalytic domain in degrading E-cadherin at 500 nM but not at a lower concentration (250 nM). These results suggest that the hemagglutinin domain of Kgp plays a role in degradation and that there is a critical threshold concentration for this activity. Taken together, these results provide evidence that the gingipains, especially Kgp, are involved in the degradation of the adherens junction of epithelial cells, which may be important in the invasion of periodontal connective tissue by P. gingivalis.

scholarly journals Actin protrusions push on E-cadherin to maintain epithelial cell-cell adhesion

2019 ◽  
Author(s):  
John Xiao He Li ◽  
Vivian W. Tang ◽  
William M. Brieher
Keyword(s):  
Cell Adhesion ◽  
Actin Polymerization ◽  
Mdck Cells ◽  
Myosin Ii ◽  
Cell Junctions ◽  
Apical Junctions ◽  
Actin Protrusions ◽  
Adhesive Contacts ◽  
E Cadherin ◽  
Cell Cell

AbstractCadherin mediated cell-cell adhesion is actin dependent, but the precise role of actin in maintaining cell-cell adhesion is not fully understood. Actin polymerization-dependent protrusive activity is required to push distally separated cells close enough together to initiate contact. Whether protrusive activity is required to maintain adhesion in confluent sheets of epithelial cells is not known. By electron microscopy as well as live cell imaging, we have identified a population of protruding actin microspikes that operate continuously near apical junctions of polarized MDCK cells. Live imaging shows that microspikes containing E-cadherin extend into gaps between E-cadherin clusters on neighboring cells while reformation of cadherin clusters across the cell-cell boundary triggers microspike withdrawal. We identify Arp2/3, EVL, and CRMP-1 as three actin assembly factors necessary for microspike formation. Depleting these factors from cells using RNAi results in myosin II-dependent unzipping of cadherin adhesive bonds. Therefore, actin polymerization-dependent protrusive activity operates continuously at cadherin cell-cell junctions to keep them shut and to prevent myosin II-dependent contractility from tearing cadherin adhesive contacts apart.

scholarly journals Epithelial self-healing is recapitulated by a 3D biomimetic E-cadherin junction

2016 ◽  
Vol 113 (51) ◽  
pp. 14698-14703 ◽  
Author(s):  
Daniel J. Cohen ◽  
Martijn Gloerich ◽  
W. James Nelson
Keyword(s):  
Epithelial Cell ◽  
Vertical Surface ◽  
Stop Signal ◽  
Cell Junctions ◽  
Epithelial Tissue ◽  
Self Healing ◽  
Material Interfaces ◽  
Cell Adhesions ◽  
E Cadherin ◽  
Cell Cell

Epithelial monolayers undergo self-healing when wounded. During healing, cells collectively migrate into the wound site, and the converging tissue fronts collide and form a stable interface. To heal, migrating tissues must form cell–cell adhesions and reorganize from the front-rear polarity characteristic of cell migration to the apical-basal polarity of an epithelium. However, identifying the "stop signal" that induces colliding tissues to cease migrating and heal remains an open question. Epithelial cells form integrin-based adhesions to the basal extracellular matrix (ECM) and E-cadherin–mediated cell–cell adhesions on the orthogonal, lateral surfaces between cells. Current biological tools have been unable to probe this multicellular 3D interface to determine the stop signal. We addressed this problem by developing a unique biointerface that mimicked the 3D organization of epithelial cell adhesions. This "minimal tissue mimic" (MTM) comprised a basal ECM substrate and a vertical surface coated with purified extracellular domain of E-cadherin, and was designed for collision with the healing edge of an epithelial monolayer. Three-dimensional imaging showed that adhesions formed between cells, and the E-cadherin-coated MTM resembled the morphology and dynamics of native epithelial cell–cell junctions and induced the same polarity transition that occurs during epithelial self-healing. These results indicate that E-cadherin presented in the proper 3D context constitutes a minimum essential stop signal to induce self-healing. That the Ecad:Fc MTM stably integrated into an epithelial tissue and reduced migration at the interface suggests that this biointerface is a complimentary approach to existing tissue–material interfaces.

scholarly journals Spatial distribution of cell–cell and cell–ECM adhesions regulates force balance while main­taining E-cadherin molecular tension in cell pairs

2015 ◽  
Vol 26 (13) ◽  
pp. 2456-2465 ◽  
Author(s):  
Joo Yong Sim ◽  
Jens Moeller ◽  
Kevin C. Hart ◽  
Diego Ramallo ◽  
Viola Vogel ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Single Cells ◽  
Force Balance ◽  
Cell Junctions ◽  
Traction Forces ◽  
Cell Pair ◽  
Spread Area ◽  
Cell Spread ◽  
E Cadherin ◽  
Cell Cell

Mechanical linkage between cell–cell and cell–extracellular matrix (ECM) adhesions regulates cell shape changes during embryonic development and tissue homoeostasis. We examined how the force balance between cell–cell and cell–ECM adhesions changes with cell spread area and aspect ratio in pairs of MDCK cells. We used ECM micropatterning to drive different cytoskeleton strain energy states and cell-generated traction forces and used a Förster resonance energy transfer tension biosensor to ask whether changes in forces across cell–cell junctions correlated with E-cadherin molecular tension. We found that continuous peripheral ECM adhesions resulted in increased cell–cell and cell–ECM forces with increasing spread area. In contrast, confining ECM adhesions to the distal ends of cell–cell pairs resulted in shorter junction lengths and constant cell–cell forces. Of interest, each cell within a cell pair generated higher strain energies than isolated single cells of the same spread area. Surprisingly, E-cadherin molecular tension remained constant regardless of changes in cell–cell forces and was evenly distributed along cell–cell junctions independent of cell spread area and total traction forces. Taken together, our results showed that cell pairs maintained constant E-cadherin molecular tension and regulated total forces relative to cell spread area and shape but independently of total focal adhesion area.

scholarly journals FSGS3/CD2AP is a barbed-end capping protein that stabilizes actin and strengthens adherens junctions

2013 ◽  
Vol 203 (5) ◽  
pp. 815-833 ◽  
Author(s):  
Vivian W. Tang ◽  
William M. Brieher
Keyword(s):  
Adhesive Strength ◽  
Mdck Cells ◽  
Adherens Junction ◽  
Cell Junctions ◽  
Actin Dynamics ◽  
Permeability Barrier ◽  
Capping Protein ◽  
End Capping ◽  
Cell Cell

By combining in vitro reconstitution biochemistry with a cross-linking approach, we have identified focal segmental glomerulosclerosis 3/CD2-associated protein (FSGS3/CD2AP) as a novel actin barbed-end capping protein responsible for actin stability at the adherens junction. FSGS3/CD2AP colocalizes with E-cadherin and α-actinin-4 at the apical junction in polarized Madin-Darby canine kidney (MDCK) cells. Knockdown of FSGS3/CD2AP compromised actin stability and decreased actin accumulation at the adherens junction. Using a novel apparatus to apply mechanical stress to cell–cell junctions, we showed that knockdown of FSGS3/CD2AP compromised adhesive strength, resulting in tearing between cells and disruption of barrier function. Our results reveal a novel function of FSGS3/CD2AP and a previously unrecognized role of barbed-end capping in junctional actin dynamics. Our study underscores the complexity of actin regulation at cell–cell contacts that involves actin activators, inhibitors, and stabilizers to control adhesive strength, epithelial behavior, and permeability barrier integrity.

scholarly journals Analysis of E-Cadherin as a Tension Sensor at Epithelial Cell-Cell Junctions

2012 ◽  
Vol 102 (3) ◽  
pp. 703a
Author(s):  
Mariya N. Sorokina ◽  
Nicolas Borghi ◽  
Olga Shcherbakova ◽  
James Nelson ◽  
Alexander Dunn
Keyword(s):  
Epithelial Cell ◽  
Cell Junctions ◽  
Tension Sensor ◽  
E Cadherin ◽  
Cell Cell

scholarly journals Actin protrusions push at apical junctions to maintain E-cadherin adhesion

2019 ◽  
Vol 117 (1) ◽  
pp. 432-438 ◽  
Author(s):  
John Xiao He Li ◽  
Vivian W. Tang ◽  
William M. Brieher
Keyword(s):  
Cell Adhesion ◽  
Actin Polymerization ◽  
Mdck Cells ◽  
Myosin Ii ◽  
Cell Junctions ◽  
Apical Junctions ◽  
Actin Protrusions ◽  
Adhesive Contacts ◽  
E Cadherin ◽  
Cell Cell

Cadherin-mediated cell–cell adhesion is actin-dependent, but the precise role of actin in maintaining cell–cell adhesion is not fully understood. Actin polymerization-dependent protrusive activity is required to push distally separated cells close enough to initiate contact. Whether protrusive activity is required to maintain adhesion in confluent sheets of epithelial cells is not known. By electron microscopy as well as live cell imaging, we have identified a population of protruding actin microspikes that operate continuously near apical junctions of polarized Madin-Darby canine kidney (MDCK) cells. Live imaging shows that microspikes containing E-cadherin extend into gaps between E-cadherin clusters on neighboring cells, while reformation of cadherin clusters across the cell–cell boundary correlates with microspike withdrawal. We identify Arp2/3, EVL, and CRMP-1 as 3 actin assembly factors necessary for microspike formation. Depleting these factors from cells using RNA interference (RNAi) results in myosin II-dependent unzipping of cadherin adhesive bonds. Therefore, actin polymerization-dependent protrusive activity operates continuously at cadherin cell–cell junctions to keep them shut and to prevent myosin II-dependent contractility from tearing cadherin adhesive contacts apart.

scholarly journals PAR1b Promotes Cell–Cell Adhesion and Inhibits Dishevelled-mediated Transformation of Madin-Darby Canine Kidney Cells

2006 ◽  
Vol 17 (8) ◽  
pp. 3345-3355 ◽  
Author(s):  
Maya Elbert ◽  
David Cohen ◽  
Anne Müsch
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cells ◽  
Wnt Signaling ◽  
Mdck Cells ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin ◽  
Cell Cell

Mammalian Par1 is a family of serine/threonine kinases comprised of four homologous isoforms that have been associated with tumor suppression and differentiation of epithelial and neuronal cells, yet little is known about their cellular functions. In polarizing kidney epithelial (Madin-Darby canine kidney [MDCK]) cells, the Par1 isoform Par1b/MARK2/EMK1 promotes the E-cadherin–dependent compaction, columnarization, and cytoskeletal organization characteristic of differentiated columnar epithelia. Here, we identify two functions of Par1b that likely contribute to its role as a tumor suppressor in epithelial cells. 1) The kinase promotes cell–cell adhesion and resistance of E-cadherin to extraction by nonionic detergents, a measure for the association of the E-cadherin cytoplasmic domain with the actin cytoskeleton, which is critical for E-cadherin function. 2) Par1b attenuates the effect of Dishevelled (Dvl) expression, an inducer of wnt signaling that causes transformation of epithelial cells. Although Dvl is a known Par1 substrate in vitro, we determined, after mapping the PAR1b-phosphorylation sites in Dvl, that PAR1b did not antagonize Dvl signaling by phosphorylating the wnt-signaling molecule. Instead, our data suggest that both proteins function antagonistically to regulate the assembly of functional E-cadherin–dependent adhesion complexes.

Sign in / Sign up

Export Citation Format

Share Document