scholarly journals Inhibition of a negative feedback for persistent epithelial cell–cell junction contraction by p21-activated kinase 3

2019 ◽  
Author(s):  
Hiroyuki Uechi ◽  
Erina Kuranaga
Keyword(s):  
Epithelial Cell ◽  
Negative Feedback ◽  
Myosin Ii ◽  
Cell Junction ◽  
Mechanical Forces ◽  
Tensile Forces ◽  
P21 Activated Kinase ◽  
Actin Protrusions ◽  
E Cadherin ◽  
Cell Cell

AbstractActin-mediated mechanical forces are central drivers of cellular dynamics. They generate protrusive and contractile dynamics, the latter of which are induced in concert with myosin II bundled at the site of contraction. These dynamics emerge concomitantly in tissues and even each cell; thus, the tight regulation of such bidirectional forces is important for proper cellular deformation. Here, we show that contractile dynamics can eventually disturb cell–cell junction contraction in the absence of p21-activated kinase 3 (Pak3). Upon Pak3 depletion, contractility induces the formation of abnormal actin protrusions at the shortening junctions, which reduces E-cadherin levels at adherens junctions. Such E-cadherin dilution dissociates myosin II from the contracting junctions, leading to a reduction in junctional tensile forces. Overexpressing E-cadherin restores the association of myosin II at the junctions and junction contraction. Our results suggest that contractility both induces and perturbs junction contraction and that the attenuation of such perturbations by Pak3 facilitates persistent junction shortening.

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 Distinct contributions of tensile and shear stress on E-cadherin levels during morphogenesis

2018 ◽  
Author(s):  
Girish R. Kale ◽  
Xingbo Yang ◽  
Jean-Marc Philippe ◽  
Madhav Mani ◽  
Pierre-François Lenne ◽  
...  
Keyword(s):  
Myosin Ii ◽  
Epithelial Morphogenesis ◽  
Shear Stresses ◽  
Mechanical Forces ◽  
Mechanical Tension ◽  
Tensile Forces ◽  
Axis Elongation ◽  
Persistent Cell ◽  
Adhesive Forces ◽  
E Cadherin

AbstractDuring epithelial morphogenesis, cell contacts (junctions) are constantly remodeled by mechanical forces that work against adhesive forces. E-cadherin complexes play a pivotal role in this process by providing persistent cell adhesion and by transmitting mechanical tension. In this context, it is unclear how mechanical forces affect E-cadherin adhesion and junction dynamics.During Drosophila embryo axis elongation, Myosin-II activity in the apico-medial and junctional cortex generates mechanical forces to drive junction remodeling. Here we report that the ratio between Vinculin and E-cadherin intensities acts as a ratiometric readout for these mechanical forces (load) at E-cadherin complexes. Medial Myosin-II loads E-cadherin complexes on all junctions, exerts tensile forces, and increases levels of E-cadherin. Junctional Myosin-II, on the other hand, biases the distribution of load between junctions of the same cell, exerts shear forces, and decreases the levels of E-cadherin. This work suggests distinct effects of tensile versus shear stresses on E-cadherin adhesion.

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 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 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 A molecular mechanism directly linking E-cadherin adhesion to initiation of epithelial cell surface polarity

2007 ◽  
Vol 178 (2) ◽  
pp. 323-335 ◽  
Author(s):  
Lene N. Nejsum ◽  
W. James Nelson
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cell ◽  
Cell Surface ◽  
Basolateral Membrane ◽  
Surface Polarity ◽  
Cell Contacts ◽  
Protein Receptors ◽  
Epithelial Cell Surface ◽  
E Cadherin ◽  
Cell Cell

Mechanisms involved in maintaining plasma membrane domains in fully polarized epithelial cells are known, but when and how directed protein sorting and trafficking occur to initiate cell surface polarity are not. We tested whether establishment of the basolateral membrane domain and E-cadherin–mediated epithelial cell–cell adhesion are mechanistically linked. We show that the basolateral membrane aquaporin (AQP)-3, but not the equivalent apical membrane AQP5, is delivered in post-Golgi structures directly to forming cell–cell contacts where it co-accumulates precisely with E-cadherin. Functional disruption of individual components of a putative lateral targeting patch (e.g., microtubules, the exocyst, and soluble N-ethylmaleimide–sensitive factor attachment protein receptors) did not inhibit cell–cell adhesion or colocalization of the other components with E-cadherin, but each blocked AQP3 delivery to forming cell–cell contacts. Thus, components of the lateral targeting patch localize independently of each other to cell–cell contacts but collectively function as a holocomplex to specify basolateral vesicle delivery to nascent cell–cell contacts and immediately initiate cell surface polarity.

scholarly journals CAR regulates epithelial cell junction stability through control of E-cadherin trafficking

2013 ◽  
Vol 3 (1) ◽  
Author(s):  
Penny E. Morton ◽  
Alexander Hicks ◽  
Theodoros Nastos ◽  
George Santis ◽  
Maddy Parsons
Keyword(s):  
Epithelial Cell ◽  
Cell Junction ◽  
E Cadherin

scholarly journals Restoration of Tight Junction Structure and Barrier Function by Down-Regulation of the Mitogen-activated Protein Kinase Pathway in Ras-transformed Madin-Darby Canine Kidney Cells

2000 ◽  
Vol 11 (3) ◽  
pp. 849-862 ◽  
Author(s):  
Yan-hua Chen ◽  
Qun Lu ◽  
Eveline E. Schneeberger ◽  
Daniel A. Goodenough
Keyword(s):  
Epithelial Cell ◽  
Tight Junction ◽  
Tight Junctions ◽  
Mitogen Activated Protein Kinase ◽  
Madin Darby Canine Kidney ◽  
Mitogen Activated Protein ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin ◽  
Cell Cell

In the Madin-Darby canine kidney epithelial cell line, the proteins occludin and ZO-1 are structural components of the tight junctions that seal the paracellular spaces between the cells and contribute to the epithelial barrier function. In Ras-transformed Madin-Darby canine kidney cells, occludin, claudin-1, and ZO-1 were absent from cell–cell contacts but were present in the cytoplasm, and the adherens junction protein E-cadherin was weakly expressed. After treatment of the Ras-transformed cells with the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059, which blocks the activation of mitogen-activated protein kinase (MAPK), occludin, claudin-1, and ZO-1 were recruited to the cell membrane, tight junctions were assembled, and E-cadherin protein expression was induced. Although it is generally believed that E-cadherin–mediated cell–cell adhesion is required for tight junction assembly, the recruitment of occludin to the cell–cell contact area and the restoration of epithelial cell morphology preceded the appearance of E-cadherin at cell–cell contacts. Both electron microscopy and a fourfold increase in the transepithelial electrical resistance indicated the formation of functional tight junctions after MEK1 inhibition. Moreover, inhibition of MAPK activity stabilized occludin and ZO-1 by differentially increasing their half-lives. We also found that during the process of tight junction assembly after MEK1 inhibition, tyrosine phosphorylation of occludin and ZO-1, but not claudin-1, increased significantly. Our study demonstrates that down-regulation of the MAPK signaling pathway causes the restoration of epithelial cell morphology and the assembly of tight junctions in Ras-transformed epithelial cells and that tyrosine phosphorylation of occludin and ZO-1 may play a role in some aspects of tight junction formation.

scholarly journals RhoA mediates epithelial cell shape changes via mechanosensitive endocytosis

2019 ◽  
Author(s):  
Kate E. Cavanaugh ◽  
Michael F. Staddon ◽  
Ed Munro ◽  
Shiladitya Banerjee ◽  
Margaret L. Gardel
Keyword(s):  
Experimental Data ◽  
Epithelial Cell ◽  
Membrane Trafficking ◽  
Cell Shape ◽  
Single Pulse ◽  
Cell Junction ◽  
Cell Contact ◽  
Rhoa Activity ◽  
Strain Dependent ◽  
Cell Cell

AbstractMorphogenetic movements require tight spatiotemporal control over cell-cell junction lengths. Contractile forces, acting at adherens junctions, alter cell-cell contact lengths in a cyclic fashion as a mechanical ratchet. Pulsatile RhoA activity is thought to drive ratcheting through acute periods of junction contraction followed by stabilization. Currently, we lack a mechanistic understanding of if and how RhoA activity governs junction length and subsequent cell shape within epithelia. In this study we use optogenetics to exogenously control RhoA activity in model Caco-2 epithelium. We find that at short timescales, RhoA activation drives reversible junction contraction. Sustained RhoA activity drives irreversible junction shortening but the amount of shortening saturates for a single pulse. To capture these data, we develop a vertex model modified to include strain-dependent junction length and tension remodeling. We find that, to account for experimental data, tension remodeling requires a strain-dependent threshold. Our model predicts that temporal structuring of RhoA activity allows for subsequent tension remodeling events to overcome the limited shortening within a single pulse and this is confirmed by our experimental data. We find that RhoA-mediated junction remodeling requires activities of formin and dynamin, indicating the closely inter-connected activities of contractility, E-cadherin clustering, and endocytosis. Junction length is therefore regulated by the coordinated action of RhoA-mediated contractility, membrane trafficking, and adhesion receptor remodeling. Altogether these data provide insights into the underlying molecular and biophysical mechanisms of RhoA-mediated regulation of epithelial cell shape.

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