scholarly journals Comprehensive analysis of formin localization in Xenopus epithelial cells

2019 ◽  
Vol 30 (1) ◽  
pp. 82-95 ◽  
Author(s):  
Tomohito Higashi ◽  
Rachel E. Stephenson ◽  
Ann L. Miller
Keyword(s):  
Epithelial Cells ◽  
Comprehensive Analysis ◽  
Cell Junctions ◽  
Actin Dynamics ◽  
Cell Junction ◽  
Contractile Ring ◽  
Dimerization Domain ◽  
Cellular Processes ◽  
And Function ◽  
Cell Cell

Reorganization of the actin cytoskeleton is crucial for cellular processes, including cytokinesis and cell–cell junction remodeling. Formins are conserved processive actin-polymerizing machines that regulate actin dynamics by nucleating, elongating, and bundling linear actin filaments. Because the formin family is large, with at least 15 members in vertebrates, there have not been any comprehensive studies examining formin localization and function within a common cell type. Here, we characterized the localization of all 15 formins in epithelial cells of Xenopus laevis gastrula-stage embryos. Dia1 and Dia2 localized to tight junctions, while Fhod1 and Fhod3 localized to adherens junctions. Only Dia3 strongly localized at the cytokinetic contractile ring. The Diaphanous inhibitory domain–dimerization domain (DID-DD) region of Dia1 was sufficient for Dia1 localization, and overexpression of a Dia1 DID-DD fragment competitively removed Dia1 and Dia2 from cell–cell junctions. In Dia1 DID-DD–overexpressing cells, Dia1 and Dia2 were mislocalized to the contractile ring, and cells exhibited increased cytokinesis failure. This work provides a comprehensive analysis of the localization of all 15 vertebrate formins in epithelial cells and suggests that misregulated formin localization results in epithelial cytokinesis failure.

scholarly journals Lulu2 regulates the circumferential actomyosin tensile system in epithelial cells through p114RhoGEF

2011 ◽  
Vol 195 (2) ◽  
pp. 245-261 ◽  
Author(s):  
Hiroyuki Nakajima ◽  
Takuji Tanoue
Keyword(s):  
Epithelial Cells ◽  
Cell Shape ◽  
Molecular Mechanisms ◽  
Molecular System ◽  
Apical Cell ◽  
Cell Junctions ◽  
Cellular Processes ◽  
Kinase C ◽  
Important Regulator ◽  
Cell Cell

Myosin II–driven mechanical forces control epithelial cell shape and morphogenesis. In particular, the circumferential actomyosin belt, which is located along apical cell–cell junctions, regulates many cellular processes. Despite its importance, the molecular mechanisms regulating the belt are not fully understood. In this paper, we characterize Lulu2, a FERM (4.1 protein, ezrin, radixin, moesin) domain–containing molecule homologous to Drosophila melanogaster Yurt, as an important regulator. In epithelial cells, Lulu2 is localized along apical cell–cell boundaries, and Lulu2 depletion by ribonucleic acid interference results in disorganization of the circumferential actomyosin belt. In its regulation of the belt, Lulu2 interacts with and activates p114RhoGEF, a Rho-specific guanine nucleotide exchanging factor (GEF), at apical cell–cell junctions. This interaction is negatively regulated via phosphorylation events in the FERM-adjacent domain of Lulu2 catalyzed by atypical protein kinase C. We further found that Patj, an apical cell polarity regulator, recruits p114RhoGEF to apical cell–cell boundaries via PDZ (PSD-95/Dlg/ZO-1) domain–mediated interaction. These findings therefore reveal a novel molecular system regulating the circumferential actomyosin belt in epithelial cells.

scholarly journals Enteropathogenic Escherichia coli (EPEC) Recruitment of PAR Polarity Protein Atypical PKCζ to Pedestals and Cell–Cell Contacts Precedes Disruption of Tight Junctions in Intestinal Epithelial Cells

2020 ◽  
Vol 21 (2) ◽  
pp. 527 ◽  
Author(s):  
Rocio Tapia ◽  
Sarah E. Kralicek ◽  
Gail A. Hecht
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Effector Proteins ◽  
Actin Dynamics ◽  
Intestinal Epithelial ◽  
Enteropathogenic Escherichia Coli ◽  
Sorting Nexin ◽  
And Function ◽  
Cell Cell

Enteropathogenic Escherichia coli (EPEC) uses a type three secretion system to inject effector proteins into host intestinal epithelial cells, causing diarrhea. EPEC induces the formation of pedestals underlying attached bacteria, disrupts tight junction (TJ) structure and function, and alters apico-basal polarity by redistributing the polarity proteins Crb3 and Pals1, although the mechanisms are unknown. Here we investigate the temporal relationship of PAR polarity complex and TJ disruption following EPEC infection. EPEC recruits active aPKCζ, a PAR polarity protein, to actin within pedestals and at the plasma membrane prior to disrupting TJ. The EPEC effector EspF binds the endocytic protein sorting nexin 9 (SNX9). This interaction impacts actin pedestal organization, recruitment of active aPKCζ to actin at cell–cell borders, endocytosis of JAM-A S285 and occludin, and TJ barrier function. Collectively, data presented herein support the hypothesis that EPEC-induced perturbation of TJ is a downstream effect of disruption of the PAR complex and that EspF binding to SNX9 contributes to this phenotype. aPKCζ phosphorylates polarity and TJ proteins and participates in actin dynamics. Therefore, the early recruitment of aPKCζ to EPEC pedestals and increased interaction with actin at the membrane may destabilize polarity complexes ultimately resulting in perturbation of TJ.

scholarly journals MgcRacGAP restricts active RhoA at the cytokinetic furrow and both RhoA and Rac1 at cell–cell junctions in epithelial cells

2015 ◽  
Vol 26 (13) ◽  
pp. 2439-2455 ◽  
Author(s):  
Elaina B. Breznau ◽  
Ansley C. Semack ◽  
Tomohito Higashi ◽  
Ann L. Miller
Keyword(s):  
Epithelial Cells ◽  
Rho Gtpases ◽  
Adherens Junction ◽  
Cell Junctions ◽  
Cell Junction ◽  
Cellular Functions ◽  
Dividing Cells ◽  
Junction Structure ◽  
Xenopus Laevis Embryos ◽  
Cell Cell

Localized activation of Rho GTPases is essential for multiple cellular functions, including cytokinesis and formation and maintenance of cell–cell junctions. Although MgcRacGAP (Mgc) is required for spatially confined RhoA-GTP at the equatorial cortex of dividing cells, both the target specificity of Mgc's GAP activity and the involvement of phosphorylation of Mgc at Ser-386 are controversial. In addition, Mgc's function at cell–cell junctions remains unclear. Here, using gastrula-stage Xenopus laevis embryos as a model system, we examine Mgc's role in regulating localized RhoA-GTP and Rac1-GTP in the intact vertebrate epithelium. We show that Mgc's GAP activity spatially restricts accumulation of both RhoA-GTP and Rac1-GTP in epithelial cells—RhoA at the cleavage furrow and RhoA and Rac1 at cell–cell junctions. Phosphorylation at Ser-386 does not switch the specificity of Mgc's GAP activity and is not required for successful cytokinesis. Furthermore, Mgc regulates adherens junction but not tight junction structure, and the ability to regulate adherens junctions is dependent on GAP activity and signaling via the RhoA pathway. Together these results indicate that Mgc's GAP activity down-regulates the active populations of RhoA and Rac1 at localized regions of epithelial cells and is necessary for successful cytokinesis and cell–cell junction structure.

scholarly journals The Herpes Simplex Virus gE-gI Complex Facilitates Cell-to-Cell Spread and Binds to Components of Cell Junctions

1998 ◽  
Vol 72 (11) ◽  
pp. 8933-8942 ◽  
Author(s):  
Kevin S. Dingwell ◽  
David C. Johnson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Cell Junctions ◽  
Cell Junction ◽  
Cell Contact ◽  
Junction Protein ◽  
Simplex Virus ◽  
Cell Cell

ABSTRACT The herpes simplex virus (HSV) glycoprotein complex gE-gI mediates the spread of viruses between adjacent cells, and this property is especially evident for cells that form extensive cell junctions, e.g., epithelial cells, fibroblasts, and neurons. Mutants lacking gE or gI are not compromised in their ability to enter cells as extracellular viruses. Therefore, gE-gI functions specifically in the movement of virus across cell-cell contacts and, as such, provides a molecular handle on this poorly understood process. We expressed gE-gI in human epithelial cells by using replication-defective adenovirus (Ad) vectors. gE-gI accumulated at lateral surfaces of the epithelial cells, colocalizing with the adherens junction protein β-catenin but was not found on either the apical or basal plasma membranes and did not colocalize with ZO-1, a component of tight junctions. In subconfluent monolayers, gE-gI was found at cell junctions but was absent from those lateral surfaces not in contact with another cell, as was the case for β-catenin. Similar localization of gE-gI to cell junctions was observed in HSV-infected epithelial cells. By contrast, HSV glycoprotein gD, expressed using a recombinant Ad vectors, was found primarily along the apical surfaces of cells, with little or no protein found on the basal or lateral surfaces. Expression of gE-gI without other HSV polypeptides did not cause redistribution of either ZO-1 or β-catenin or alter tight-junction functions. Together these results support a model in which gE-gI accumulates at sites of cell-cell contact by interacting with junctional components. We hypothesize that gE-gI mediates transfer of HSV across cell junctions by virtue of these interactions with cell junction components.

scholarly journals Regulation of Cell-Cell Adhesion by Abi/Diaphanous Complexes

2009 ◽  
Vol 29 (7) ◽  
pp. 1735-1748 ◽  
Author(s):  
Jae Ryun Ryu ◽  
Asier Echarri ◽  
Ran Li ◽  
Ann Marie Pendergast
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cells ◽  
Driving Force ◽  
Actin Polymerization ◽  
Binding Protein ◽  
Cell Junctions ◽  
Actin Dynamics ◽  
Binding Partner ◽  
Terminal Fragment ◽  
Cell Cell

ABSTRACT Actin polymerization provides the driving force for the formation of cell-cell junctions and is mediated by two types of actin nucleators, Arp2/3 and formins. Proteins required for coordinately linking cadherin-mediated adhesion to Arp2/3-dependent versus formin-dependent nucleation have yet to be defined. Here we show a role for Abi, the Abi-binding partner Nap1, and the Nap1-binding protein Sra1 in the regulation of cadherin-dependent adhesion. We found that Abi, which is known to interact with Wave, leading to activation of the Arp2/3 complex, is also capable of interacting with the Diaphanous (Dia)-related formins in the absence of Wave. Knockdown of Abi, Nap1, Sra1, or Dia markedly inhibited cell-cell junctions, whereas knockdown of Wave or Arp2/3 produced mild and transient phenotypes. Dia and Abi colocalized with β-catenin at cell-cell junctions. Further, Dia and Wave bound to overlapping sites on Abi1, and Wave competed with Dia for Abi1 binding. Notably, an active Dia1 C-terminal fragment that localizes to cell-cell junctions rescued the abnormal junctions induced by depletion of Abi or Nap1 in epithelial cells. These findings uncover a novel link between cadherin-mediated adhesion and the regulation of actin dynamics through the requirement for an Abi/Dia complex for the formation and stability of cell-cell junctions.

scholarly journals Out, in and back again: PtdIns(4,5)P2 regulates cadherin trafficking in epithelial morphogenesis

2009 ◽  
Vol 418 (2) ◽  
pp. 247-260 ◽  
Author(s):  
Nicholas J. Schill ◽  
Richard A. Anderson
Keyword(s):  
Epithelial Morphogenesis ◽  
Cell Junctions ◽  
Functional Protein ◽  
Regulatory Pathways ◽  
Cellular Processes ◽  
Endocytic Machinery ◽  
Cellular Phenotypes ◽  
And Function ◽  
E Cadherin ◽  
Cell Cell

The morphogenesis of epithelial cells in the tissue microenvironment depends on the regulation of the forces and structures that keep cells in contact with their neighbours. The formation of cell–cell contacts is integral to the establishment and maintenance of epithelial morphogenesis. In epithelial tissues, the misregulation of the signalling pathways that control epithelial polarization induces migratory and invasive cellular phenotypes. Many cellular processes influence cadherin targeting and function, including exocytosis, endocytosis and recycling. However, the localized generation of the lipid messenger PtdIns(4,5)P2 is emerging as a fundamental signal controlling all of these processes. The PtdIns(4,5)P2-generating enzymes, PIPKs (phosphatidylinositol phosphate kinases) are therefore integral to these pathways. By the spatial and temporal targeting of PIPKs via the actions of its functional protein associates, PtdIns(4,5)P2 is generated at discrete cellular locales to provide the cadherin-trafficking machinery with its required lipid messenger. In the present review, we discuss the involvement of PtdIns(4,5)P2 and the PIPKs in the regulation of the E-cadherin (epithelial cadherin) exocytic and endocytic machinery, the modulation of actin structures at sites of adhesion, and the direction of cellular pathways which determine the fate of E-cadherin and cell–cell junctions. Recent work is also described that has defined phosphoinositide-mediated E-cadherin regulatory pathways by the use of organismal models.

Phosphatidylinositol-4-phosphate 5-kinase γ is associated with cell–cell junction in A431 epithelial cells

2005 ◽  
Vol 29 (7) ◽  
pp. 514-520 ◽  
Author(s):  
C AKIYAMA ◽  
N SHINOZAKINARIKAWA ◽  
T KITAZAWA ◽  
T HAMAKUBO ◽  
T KODAMA ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Junction ◽  
Phosphatidylinositol 4 ◽  
Cell Cell

scholarly journals PKM2 regulates endothelial cell junction dynamics and angiogenesis via ATP production

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jesús Gómez-Escudero ◽  
Cristina Clemente ◽  
Diego García-Weber ◽  
Rebeca Acín-Pérez ◽  
Jaime Millán ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Inflammatory Disease ◽  
Chronic Inflammatory Disease ◽  
Cell Junctions ◽  
Cell Junction ◽  
Collective Migration ◽  
Sprouting Angiogenesis ◽  
Cell Cell

Abstract Angiogenesis, the formation of new blood vessels from pre-existing ones, occurs in pathophysiological contexts such as wound healing, cancer, and chronic inflammatory disease. During sprouting angiogenesis, endothelial tip and stalk cells coordinately remodel their cell-cell junctions to allow collective migration and extension of the sprout while maintaining barrier integrity. All these processes require energy, and the predominant ATP generation route in endothelial cells is glycolysis. However, it remains unclear how ATP reaches the plasma membrane and intercellular junctions. In this study, we demonstrate that the glycolytic enzyme pyruvate kinase 2 (PKM2) is required for sprouting angiogenesis in vitro and in vivo through the regulation of endothelial cell-junction dynamics and collective migration. We show that PKM2-silencing decreases ATP required for proper VE-cadherin internalization/traffic at endothelial cell-cell junctions. Our study provides fresh insight into the role of ATP subcellular compartmentalization in endothelial cells during angiogenesis. Since manipulation of EC glycolysis constitutes a potential therapeutic intervention route, particularly in tumors and chronic inflammatory disease, these findings may help to refine the targeting of endothelial glycolytic activity in disease.

scholarly journals Deciliation Is Associated with Dramatic Remodeling of Epithelial Cell Junctions and Surface Domains

2009 ◽  
Vol 20 (1) ◽  
pp. 102-113 ◽  
Author(s):  
Christian E. Overgaard ◽  
Kaitlin M. Sanzone ◽  
Krystle S. Spiczka ◽  
David R. Sheff ◽  
Alexander Sandra ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Primary Cilia ◽  
Mdck Cells ◽  
Cell Junctions ◽  
Apical Trafficking ◽  
Epithelial Remodeling ◽  
Surface Domains ◽  
And Function ◽  
Mediated Reduction ◽  
Junction Proteins

Stress-induced shedding of motile cilia (autotomy) has been documented in diverse organisms and likely represents a conserved cellular reaction. However, little is known about whether primary cilia are shed from mammalian epithelial cells and what impact deciliation has on polarized cellular organization. We show that several chemically distinct agents trigger autotomy in epithelial cells. Surprisingly, deciliation is associated with a significant, but reversible increase in transepithelial resistance. This reflects substantial reductions in tight junction proteins associated with “leaky” nephron segments (e.g., claudin-2). At the same time, apical trafficking of gp80/clusterin and gp114/CEACAM becomes randomized, basal-lateral delivery of Na,K-ATPase is reduced, and expression of the nonciliary apical protein gp135/podocalyxin is greatly decreased. However, ciliogenesis-impaired MDCK cells do not undergo continual junction remodeling, and mature cilia are not required for autotomy-associated remodeling events. Deciliation and epithelial remodeling may be mechanistically linked processes, because RNAi-mediated reduction of Exocyst subunit Sec6 inhibits ciliary shedding and specifically blocks deciliation-associated down-regulation of claudin-2 and gp135. We propose that ciliary autotomy represents a signaling pathway that impacts the organization and function of polarized epithelial cells.

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