scholarly journals Epithelial junction formation requires confinement of Cdc42 activity by a novel SH3BP1 complex

2012 ◽  
Vol 198 (4) ◽  
pp. 677-693 ◽  
Author(s):  
Ahmed Elbediwy ◽  
Ceniz Zihni ◽  
Stephen J. Terry ◽  
Peter Clark ◽  
Karl Matter ◽  
...  
Keyword(s):  
Dynamic Control ◽  
Scaffolding Protein ◽  
Cell Junction ◽  
Membrane Remodeling ◽  
Filamentous Actin ◽  
Capping Protein ◽  
Junction Formation ◽  
Guanosine Triphosphatase ◽  
Epithelial Junction ◽  
Cell Cell

Epithelial cell–cell adhesion and morphogenesis require dynamic control of actin-driven membrane remodeling. The Rho guanosine triphosphatase (GTPase) Cdc42 regulates sequential molecular processes during cell–cell junction formation; hence, mechanisms must exist that inactivate Cdc42 in a temporally and spatially controlled manner. In this paper, we identify SH3BP1, a GTPase-activating protein for Cdc42 and Rac, as a regulator of junction assembly and epithelial morphogenesis using a functional small interfering ribonucleic acid screen. Depletion of SH3BP1 resulted in loss of spatial control of Cdc42 activity, stalled membrane remodeling, and enhanced growth of filopodia. SH3BP1 formed a complex with JACOP/paracingulin, a junctional adaptor, and CD2AP, a scaffolding protein; both were required for normal Cdc42 signaling and junction formation. The filamentous actin–capping protein CapZ also associated with the SH3BP1 complex and was required for control of actin remodeling. Epithelial junction formation and morphogenesis thus require a dual activity complex, containing SH3BP1 and CapZ, that is recruited to sites of active membrane remodeling to guide Cdc42 signaling and cytoskeletal dynamics.

scholarly journals PLEKHG4B enables actin cytoskeletal remodeling during epithelial cell-cell junction formation

2020 ◽  
pp. jcs.249078
Author(s):  
Komaki Ninomiya ◽  
Kai Ohta ◽  
Kazunari Yamashita ◽  
Kensaku Mizuno ◽  
Kazumasa Ohashi
Keyword(s):  
Epithelial Cell ◽  
A549 Cells ◽  
Cell Junctions ◽  
Cell Junction ◽  
Nucleotide Exchange ◽  
Actin Bundles ◽  
Rhoa Activity ◽  
Cytoskeletal Remodeling ◽  
Junction Formation ◽  
Cell Cell

Cell-cell junction formation requires actin cytoskeletal remodeling. Here we show that PLEKHG4B, a Rho-guanine nucleotide exchange factor (Rho-GEF), plays a crucial role in epithelial cell-cell junction formation. Knockdown of PLEKHG4B decreased Cdc42 activity and tended to increase RhoA activity in A549 cells. A549 monolayer cells showed 'closed junctions' with closely packed actin bundles along the cell-cell contacts, but PLEKHG4B knockdown suppressed closed junction formation and exhibited 'open junctions' with split actin bundles located away from the cell-cell boundary. In calcium-switch assays, PLEKHG4B knockdown delayed the conversion of open junctions to closed junctions and β-catenin accumulation at cell-cell junctions. Further, PLEKHG4B knockdown abrogated the reduction in myosin activity normally seen in the later stage of junction formation. The aberrant myosin activation and impairments in closed junction formation in PLEKHG4B-knockdown cells were reverted by ROCK inhibition or LARG/PDZ-RhoGEF knockdown. These results suggest that PLEKHG4B enables actin remodeling during epithelial cell-cell junction maturation, probably by reducing myosin activity in the later stage of junction formation, through suppressing LARG/PDZ-RhoGEF and RhoA-ROCK activities. We also showed that annexin-A2 participates in PLEKHG4B localization to cell-cell junctions.

scholarly journals The cytoskeletal mechanisms of cell–cell junction formation in endothelial cells

2012 ◽  
Vol 23 (2) ◽  
pp. 310-323 ◽  
Author(s):  
Matthew K. Hoelzle ◽  
Tatyana Svitkina
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Adherens Junctions ◽  
Myosin Ii ◽  
Vital Role ◽  
Cell Junction ◽  
Bridge Formation ◽  
Actin Organization ◽  
Junction Formation ◽  
Cell Cell

The actin cytoskeleton and associated proteins play a vital role in cell–cell adhesion. However, the procedure by which cells establish adherens junctions remains unclear. We investigated the dynamics of cell–cell junction formation and the corresponding architecture of the underlying cytoskeleton in cultured human umbilical vein endothelial cells. We show that the initial interaction between cells is mediated by protruding lamellipodia. On their retraction, cells maintain contact through thin bridges formed by filopodia-like protrusions connected by VE-cadherin–rich junctions. Bridges share multiple features with conventional filopodia, such as an internal actin bundle associated with fascin along the length and vasodilator-stimulated phosphoprotein at the tip. It is striking that, unlike conventional filopodia, transformation of actin organization from the lamellipodial network to filopodial bundle during bridge formation occurs in a proximal-to-distal direction and is accompanied by recruitment of fascin in the same direction. Subsequently, bridge bundles recruit nonmuscle myosin II and mature into stress fibers. Myosin II activity is important for bridge formation and accumulation of VE-cadherin in nascent adherens junctions. Our data reveal a mechanism of cell–cell junction formation in endothelial cells using lamellipodia as the initial protrusive contact, subsequently transforming into filopodia-like bridges connected through adherens junctions. Moreover, a novel lamellipodia-to-filopodia transition is used in this context.

scholarly journals Accumulation of a microtubule-binding protein, pp170, at desmosomal plaques

1992 ◽  
Vol 117 (4) ◽  
pp. 813-824 ◽  
Author(s):  
IU Wacker ◽  
JE Rickard ◽  
JR De Mey ◽  
TE Kreis
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Polarity ◽  
Mdck Cells ◽  
Binding Protein ◽  
Cell Junction ◽  
Microtubule Binding ◽  
Epithelial Cell Polarity ◽  
Junction Formation ◽  
Cell Cell

The establishment of epithelial cell polarity correlates with the formation of specialized cell-cell junctions and striking changes in the organization of microtubules. A significant fraction of the microtubules in MDCK cells become stabilized, noncentrosomally organized, and arranged in longitudinal bundles in the apical-basal axis. This correlation suggests a functional link between cell-cell junction formation and control of microtubule organization. We have followed the distribution of pp170, a recently described microtubule-binding protein, during establishment of epithelial cell polarity. This protein shows the typical patchy distribution along microtubules in subconfluent fibroblasts and epithelial cells, often associated with the peripheral ends of a subpopulation of microtubules. In contrast to its localization in confluent fibroblasts (A72) and HeLa cells, however, pp170 accumulates in patches delineating the regions of cell-cell contacts in confluent polarizing epithelial cells (MDCK and Caco-2). Double immunolocalization with antibodies specific for cell-cell junction proteins, confocal microscopy, and immunoelectron microscopy on polarized MDCK cells suggest that pp170 accumulates at desmosomal plaques. Furthermore, microtubules and desmosomes are found in close contact. Maintenance of the desmosomal association of pp170 is dependent on intact microtubules in 3-d-old, but not in 1-d-old MDCK cell cultures. This suggests a regulated interaction between microtubules and desmosomes and a role for pp170 in the control of changes in the properties of microtubules induced by epithelial cell-cell junction formation.

scholarly journals Rap1: a key regulator in cell-cell junction formation

2006 ◽  
Vol 120 (1) ◽  
pp. 17-22 ◽  
Author(s):  
M. R. H. Kooistra ◽  
N. Dube ◽  
J. L. Bos
Keyword(s):  
Cell Junction ◽  
Junction Formation ◽  
Cell Cell

scholarly journals Golgi-associated cPLA2α Regulates Endothelial Cell–Cell Junction Integrity by Controlling the Trafficking of Transmembrane Junction Proteins

2009 ◽  
Vol 20 (19) ◽  
pp. 4225-4234 ◽  
Author(s):  
Elsa Regan-Klapisz ◽  
Vincent Krouwer ◽  
Miriam Langelaar-Makkinje ◽  
Laxman Nallan ◽  
Michael Gelb ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Intracellular Trafficking ◽  
Adherens Junction ◽  
Cell Junctions ◽  
Cell Junction ◽  
Tight Junction Formation ◽  
Junction Formation ◽  
Junction Proteins ◽  
Cell Cell

In endothelial cells specifically, cPLA2α translocates from the cytoplasm to the Golgi complex in response to cell confluence. Considering the link between confluence and cell–cell junction formation, and the emerging role of cPLA2α in intracellular trafficking, we tested whether Golgi-associated cPLA2α is involved in the trafficking of junction proteins. Here, we show that the redistribution of cPLA2α from the cytoplasm to the Golgi correlates with adherens junction maturation and occurs before tight junction formation. Disruption of adherens junctions using a blocking anti-VE-cadherin antibody reverses the association of cPLA2α with the Golgi. Silencing of cPLA2α and inhibition of cPLA2α enzymatic activity using various inhibitors result in the diminished presence of the transmembrane junction proteins VE-cadherin, occludin, and claudin-5 at cell–cell contacts, and in their accumulation at the Golgi. Altogether, our data support the idea that VE-cadherin triggers the relocation of cPLA2α to the Golgi and that in turn, Golgi-associated cPLA2α regulates the transport of transmembrane junction proteins through or from the Golgi, thereby controlling the integrity of endothelial cell–cell junctions.

scholarly journals M-Ras/Shoc2 signaling modulates E-cadherin turnover and cell–cell adhesion during collective cell migration

2019 ◽  
Vol 116 (9) ◽  
pp. 3536-3545 ◽  
Author(s):  
Pradeep Kota ◽  
Elizabeth M. Terrell ◽  
Daniel A. Ritt ◽  
Christine Insinna ◽  
Christopher J. Westlake ◽  
...  
Keyword(s):  
Cell Migration ◽  
Dynamic Control ◽  
Cell Movement ◽  
Cell Junction ◽  
Collective Cell Migration ◽  
Dynamic Regulation ◽  
P120 Catenin ◽  
Mcf10a Cells ◽  
E Cadherin ◽  
Cell Cell

Collective cell migration is required for normal embryonic development and contributes to various biological processes, including wound healing and cancer cell invasion. The M-Ras GTPase and its effector, the Shoc2 scaffold, are proteins mutated in the developmental RASopathy Noonan syndrome, and, here, we report that activated M-Ras recruits Shoc2 to cell surface junctions where M-Ras/Shoc2 signaling contributes to the dynamic regulation of cell–cell junction turnover required for collective cell migration. MCF10A cells expressing the dominant-inhibitory M-RasS27N variant or those lacking Shoc2 exhibited reduced junction turnover and were unable to migrate effectively as a group. Through further depletion/reconstitution studies, we found that M-Ras/Shoc2 signaling contributes to junction turnover by modulating the E-cadherin/p120-catenin interaction and, in turn, the junctional expression of E-cadherin. The regulatory effect of the M-Ras/Shoc2 complex was mediated at least in part through the phosphoregulation of p120-catenin and required downstream ERK cascade activation. Strikingly, cells rescued with the Noonan-associated, myristoylated-Shoc2 mutant (Myr-Shoc2) displayed a gain-of-function (GOF) phenotype, with the cells exhibiting increased junction turnover and reduced E-cadherin/p120-catenin binding and migrating as a faster but less cohesive group. Consistent with these results, Noonan-associated C-Raf mutants that bypass the need for M-Ras/Shoc2 signaling exhibited a similar GOF phenotype when expressed in Shoc2-depleted MCF10A cells. Finally, expression of the Noonan-associated Myr-Shoc2 or C-Raf mutants, but not their WT counterparts, induced gastrulation defects indicative of aberrant cell migration in zebrafish embryos, further demonstrating the function of the M-Ras/Shoc2/ERK cascade signaling axis in the dynamic control of coordinated cell movement.

scholarly journals Synaptopodin couples epithelial contractility to α-actinin-4–dependent junction maturation

2015 ◽  
Vol 211 (2) ◽  
pp. 407-434 ◽  
Author(s):  
Nivetha Kannan ◽  
Vivian W. Tang
Keyword(s):  
Control Process ◽  
Mechanical Force ◽  
Cell Junction ◽  
Junctional Complex ◽  
Permeability Barrier ◽  
Dependent Manner ◽  
Upstream Regulator ◽  
Adhesive Contacts ◽  
Epithelial Junction ◽  
Cell Cell

The epithelial junction experiences mechanical force exerted by endogenous actomyosin activities and from interactions with neighboring cells. We hypothesize that tension generated at cell–cell adhesive contacts contributes to the maturation and assembly of the junctional complex. To test our hypothesis, we used a hydraulic apparatus that can apply mechanical force to intercellular junction in a confluent monolayer of cells. We found that mechanical force induces α-actinin-4 and actin accumulation at the cell junction in a time- and tension-dependent manner during junction development. Intercellular tension also induces α-actinin-4–dependent recruitment of vinculin to the cell junction. In addition, we have identified a tension-sensitive upstream regulator of α-actinin-4 as synaptopodin. Synaptopodin forms a complex containing α-actinin-4 and β-catenin and interacts with myosin II, indicating that it can physically link adhesion molecules to the cellular contractile apparatus. Synaptopodin depletion prevents junctional accumulation of α-actinin-4, vinculin, and actin. Knockdown of synaptopodin and α-actinin-4 decreases the strength of cell–cell adhesion, reduces the monolayer permeability barrier, and compromises cellular contractility. Our findings underscore the complexity of junction development and implicate a control process via tension-induced sequential incorporation of junctional components.

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