scholarly journals Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1

2015 ◽  
Vol 209 (3) ◽  
pp. 367-376 ◽  
Author(s):  
Katharina Grikscheit ◽  
Tanja Frank ◽  
Ying Wang ◽  
Robert Grosse
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cells ◽  
Actin Polymerization ◽  
De Novo ◽  
Early Stage ◽  
Adhesion Formation ◽  
Actin Dynamics ◽  
Cell Contact ◽  
Actin Assembly ◽  
Cell Cell

Epithelial integrity is vitally important, and its deregulation causes early stage cancer. De novo formation of an adherens junction (AJ) between single epithelial cells requires coordinated, spatial actin dynamics, but the mechanisms steering nascent actin polymerization for cell–cell adhesion initiation are not well understood. Here we investigated real-time actin assembly during daughter cell–cell adhesion formation in human breast epithelial cells in 3D environments. We identify formin-like 2 (FMNL2) as being specifically required for actin assembly and turnover at newly formed cell–cell contacts as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cell–cell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1.

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 α-Actinin-4/FSGS1 is required for Arp2/3-dependent actin assembly at the adherens junction

2012 ◽  
Vol 196 (1) ◽  
pp. 115-130 ◽  
Author(s):  
Vivian W. Tang ◽  
William M. Brieher
Keyword(s):  
De Novo ◽  
Adherens Junction ◽  
Dominant Negative ◽  
Cell Junctions ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Vitro Assay ◽  
Apical Junctions ◽  
Cell Cell

We have developed an in vitro assay to study actin assembly at cadherin-enriched cell junctions. Using this assay, we demonstrate that cadherin-enriched junctions can polymerize new actin filaments but cannot capture preexisting filaments, suggesting a mechanism involving de novo synthesis. In agreement with this hypothesis, inhibition of Arp2/3-dependent nucleation abolished actin assembly at cell–cell junctions. Reconstitution biochemistry using the in vitro actin assembly assay identified α-actinin-4/focal segmental glomerulosclerosis 1 (FSGS1) as an essential factor. α-Actinin-4 specifically localized to sites of actin incorporation on purified membranes and at apical junctions in Madin–Darby canine kidney cells. Knockdown of α-actinin-4 decreased total junctional actin and inhibited actin assembly at the apical junction. Furthermore, a point mutation of α-actinin-4 (K255E) associated with FSGS failed to support actin assembly and acted as a dominant negative to disrupt actin dynamics at junctional complexes. These findings demonstrate that α-actinin-4 plays an important role in coupling actin nucleation to assembly at cadherin-based cell–cell adhesive contacts.

scholarly journals Surface apposition and multiple cell contacts promote myoblast fusion in Drosophila flight muscles

2015 ◽  
Vol 211 (1) ◽  
pp. 191-203 ◽  
Author(s):  
Nagaraju Dhanyasi ◽  
Dagan Segal ◽  
Eyal Shimoni ◽  
Vera Shinder ◽  
Ben-Zion Shilo ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Actin Polymerization ◽  
Cell Types ◽  
Myoblast Fusion ◽  
Cell Contact ◽  
Minimal Distance ◽  
Multiple Cell ◽  
Flight Muscles ◽  
Cytoskeletal Elements ◽  
Cell Cell

Fusion of individual myoblasts to form multinucleated myofibers constitutes a widely conserved program for growth of the somatic musculature. We have used electron microscopy methods to study this key form of cell–cell fusion during development of the indirect flight muscles (IFMs) of Drosophila melanogaster. We find that IFM myoblast–myotube fusion proceeds in a stepwise fashion and is governed by apparent cross talk between transmembrane and cytoskeletal elements. Our analysis suggests that cell adhesion is necessary for bringing myoblasts to within a minimal distance from the myotubes. The branched actin polymerization machinery acts subsequently to promote tight apposition between the surfaces of the two cell types and formation of multiple sites of cell–cell contact, giving rise to nascent fusion pores whose expansion establishes full cytoplasmic continuity. Given the conserved features of IFM myogenesis, this sequence of cell interactions and membrane events and the mechanistic significance of cell adhesion elements and the actin-based cytoskeleton are likely to represent general principles of the myoblast fusion process.

scholarly journals Localized zones of Rho and Rac activities drive initiation and expansion of epithelial cell–cell adhesion

2007 ◽  
Vol 178 (3) ◽  
pp. 517-527 ◽  
Author(s):  
Soichiro Yamada ◽  
W. James Nelson
Keyword(s):  
Cell Adhesion ◽  
Actin Filaments ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
De Novo ◽  
Actin Dynamics ◽  
Lateral Expansion ◽  
Actomyosin Contractility ◽  
Spatiotemporal Coordination ◽  
Cell Cell

Spatiotemporal coordination of cell–cell adhesion involving lamellipodial interactions, cadherin engagement, and the lateral expansion of the contact is poorly understood. Using high-resolution live-cell imaging, biosensors, and small molecule inhibitors, we investigate how Rac1 and RhoA regulate actin dynamics during de novo contact formation between pairs of epithelial cells. Active Rac1, the Arp2/3 complex, and lamellipodia are initially localized to de novo contacts but rapidly diminish as E-cadherin accumulates; further rounds of activation and down-regulation of Rac1 and Arp2/3 occur at the contacting membrane periphery, and this cycle repeats as a restricted membrane zone that moves outward with the expanding contact. The cortical bundle of actin filaments dissolves beneath the expanding contacts, leaving actin bundles at the contact edges. RhoA and actomyosin contractility are activated at the contact edges and are required to drive expansion and completion of cell–cell adhesion. We show that zones of Rac1 and lamellipodia activity and of RhoA and actomyosin contractility are restricted to the periphery of contacting membranes and together drive initiation, expansion, and completion of cell–cell adhesion.

scholarly journals Novel Kelch-like Protein, KLEIP, Is Involved in Actin Assembly at Cell-Cell Contact Sites of Madin-Darby Canine Kidney Cells

2004 ◽  
Vol 15 (3) ◽  
pp. 1172-1184 ◽  
Author(s):  
Takahiko Hara ◽  
Hiroshi Ishida ◽  
Razi Raziuddin ◽  
Stephan Dorkhom ◽  
Keiju Kamijo ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Contact ◽  
Kidney Cells ◽  
Actin Assembly ◽  
Madin Darby Canine Kidney ◽  
Contact Sites ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin ◽  
Cell Cell

Dynamic rearrangements of cell-cell adhesion underlie a diverse range of physiological processes, but their precise molecular mechanisms are still obscure. Thus, identification of novel players that are involved in cell-cell adhesion would be important. We isolated a human kelch-related protein, Kelch-like ECT2 interacting protein (KLEIP), which contains the broad-complex, tramtrack, bric-a-brac (BTB)/poxvirus, zinc finger (POZ) motif and six-tandem kelch repeats. KLEIP interacted with F-actin and was concentrated at cell-cell contact sites of Madin-Darby canine kidney cells, where it colocalized with F-actin. Interestingly, this localization took place transiently during the induction of cell-cell contact and was not seen at mature junctions. KLEIP recruitment and actin assembly were induced around E-cadherin–coated beads placed on cell surfaces. The actin depolymerizing agent cytochalasin B inhibited this KLEIP recruitment around E-cadherin–coated beads. Moreover, constitutively active Rac1 enhanced the recruitment of KLEIP as well as F-actin to the adhesion sites. These observations strongly suggest that KLEIP is localized on actin filaments at the contact sites. We also found that N-terminal half of KLEIP, which lacks the actin-binding site and contains the sufficient sequence for the localization at the cell-cell contact sites, inhibited constitutively active Rac1-induced actin assembly at the contact sites. We propose that KLEIP is involved in Rac1-induced actin organization during cell-cell contact in Madin-Darby canine kidney cells.

Microtubule dynamics and Rac-1 signaling independently regulate barrier function in lung epithelial cells

2007 ◽  
Vol 293 (5) ◽  
pp. L1321-L1331 ◽  
Author(s):  
Magdalena J. Lorenowicz ◽  
Mar Fernandez-Borja ◽  
Anne-Marieke D. van Stalborch ◽  
Marian A. J. A. van Sterkenburg ◽  
Pieter S. Hiemstra ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cells ◽  
Barrier Function ◽  
Lung Epithelial Cells ◽  
Cell Junctions ◽  
Epithelial Barrier ◽  
Cell Contact ◽  
Epithelial Barrier Function ◽  
Lung Epithelial ◽  
Cell Cell

Cadherin-mediated cell-cell adhesion controls the morphology and function of epithelial cells and is a critical component of the pathology of chronic inflammatory disorders. Dynamic interactions between cadherins and the actin cytoskeleton are required for stable cell-cell contact. Besides actin, microtubules also target intercellular, cadherin-based junctions and contribute to their formation and stability. Here, we studied the role of microtubules in conjunction with Rho-like GTPases in the regulation of lung epithelial barrier function using real-time monitoring of transepithelial electrical resistance. Unexpectedly, we found that disruption of microtubules promotes epithelial cell-cell adhesion. This increase in epithelial barrier function is accompanied by the accumulation of β-catenin at cell-cell junctions, as detected by immunofluorescence. Moreover, we found that the increase in cell-cell contact, induced by microtubule depolymerization, requires signaling through a RhoA/Rho kinase pathway. The Rac-1 GTPase counteracts this pathway, because inhibition of Rac-1 signaling rapidly promotes epithelial barrier function, in a microtubule- and RhoA-independent fashion. Together, our data suggest that microtubule-RhoA-mediated signaling and Rac-1 control lung epithelial integrity through counteracting independent pathways.

scholarly journals Adenovirus E4orf4 Hijacks Rho GTPase-dependent Actin Dynamics to Kill Cells: A Role for Endosome-associated Actin Assembly

2006 ◽  
Vol 17 (7) ◽  
pp. 3329-3344 ◽  
Author(s):  
Amélie Robert ◽  
Nicolas Smadja-Lamère ◽  
Marie-Claude Landry ◽  
Claudia Champagne ◽  
Ryan Petrie ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Actin Polymerization ◽  
Rho Gtpase ◽  
De Novo ◽  
Strong Support ◽  
Src Family Kinases ◽  
Actin Dynamics ◽  
Death Process ◽  
Actin Assembly ◽  
Orf4 Protein

The adenovirus early region 4 ORF4 protein (E4orf4) triggers a novel death program that bypasses classical apoptotic pathways in human cancer cells. Deregulation of the cell cytoskeleton is a hallmark of E4orf4 killing that relies on Src family kinases and E4orf4 phosphorylation. However, the cytoskeletal targets of E4orf4 and their role in the death process are unknown. Here, we show that E4orf4 translocates to cytoplasmic sites and triggers the assembly of a peculiar juxtanuclear actin–myosin network that drives polarized blebbing and nuclear shrinkage. We found that E4orf4 activates the myosin II motor and triggers de novo actin polymerization in the perinuclear region, promoting endosomes recruitment to the sites of actin assembly. E4orf4-induced actin dynamics requires interaction with Src family kinases and involves a spatial regulation of the Rho GTPases pathways Cdc42/N-Wasp, RhoA/Rho kinase, and Rac1, which make distinct contributions. Remarkably, activation of the Rho GTPases is required for induction of apoptotic-like cell death. Furthermore, inhibition of actin dynamics per se dramatically impairs E4orf4 killing. This work provides strong support for a causal role for endosome-associated actin dynamics in E4orf4 killing and in the regulation of cancer cell fate.

scholarly journals Cell–Cell Adhesion and Myosin Activity Regulate Cortical Actin Assembly in Mammary Gland Epithelium on Concaved Surface

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 813
Author(s):  
Wei-Hung Jung ◽  
Khalid Elawad ◽  
Sung Hoon Kang ◽  
Yun Chen
Keyword(s):  
Cell Adhesion ◽  
Mammary Gland ◽  
Cellular Level ◽  
Cell Contact ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Cell Behaviors ◽  
Curvature Sensing ◽  
Dependent Cell ◽  
Cell Cell

It has been demonstrated that geometry can affect cell behaviors. Though curvature-sensitive proteins at the nanoscale are studied, it is unclear how cells sense curvature at the cellular and multicellular levels. To characterize and determine the mechanisms of curvature-dependent cell behaviors, we grow cells on open channels of the 60-µm radius. We found that cortical F-actin is 1.2-fold more enriched in epithelial cells grown on the curved surface compared to the flat control. We observed that myosin activity is required to promote cortical F-actin formation. Furthermore, cell–cell contact was shown to be indispensable for curvature-dependent cortical actin assembly. Our results indicate that the actomyosin network coupled with adherens junctions is involved in curvature-sensing at the multi-cellular level.

scholarly journals αE-catenin regulates actin dynamics independently of cadherin-mediated cell–cell adhesion

2010 ◽  
Vol 189 (2) ◽  
pp. 339-352 ◽  
Author(s):  
Jacqueline M. Benjamin ◽  
Adam V. Kwiatkowski ◽  
Changsong Yang ◽  
Farida Korobova ◽  
Sabine Pokutta ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Actin Polymerization ◽  
Membrane Dynamics ◽  
Actin Dynamics ◽  
Filamentous Actin ◽  
Vitro Binding ◽  
Adhesion Complex ◽  
E Cadherin ◽  
Cell Cell

αE-catenin binds the cell–cell adhesion complex of E-cadherin and β-catenin (β-cat) and regulates filamentous actin (F-actin) dynamics. In vitro, binding of αE-catenin to the E-cadherin–β-cat complex lowers αE-catenin affinity for F-actin, and αE-catenin alone can bind F-actin and inhibit Arp2/3 complex–mediated actin polymerization. In cells, to test whether αE-catenin regulates actin dynamics independently of the cadherin complex, the cytosolic αE-catenin pool was sequestered to mitochondria without affecting overall levels of αE-catenin or the cadherin–catenin complex. Sequestering cytosolic αE-catenin to mitochondria alters lamellipodia architecture and increases membrane dynamics and cell migration without affecting cell–cell adhesion. In contrast, sequestration of cytosolic αE-catenin to the plasma membrane reduces membrane dynamics. These results demonstrate that the cytosolic pool of αE-catenin regulates actin dynamics independently of cell–cell adhesion.

scholarly journals Changes in E-cadherin rigidity sensing regulate cell adhesion

2017 ◽  
Vol 114 (29) ◽  
pp. E5835-E5844 ◽  
Author(s):  
Caitlin Collins ◽  
Aleksandra K. Denisin ◽  
Beth L. Pruitt ◽  
W. James Nelson
Keyword(s):  
Cell Adhesion ◽  
Signaling Molecules ◽  
Membrane Dynamics ◽  
Cell Contact ◽  
Adhesion Assay ◽  
Cell Periphery ◽  
Actin Organization ◽  
Rigidity Sensing ◽  
E Cadherin ◽  
Cell Cell

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin–dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell–cell adhesion assay and live cell imaging of cell–cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell–cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell–cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell–cell adhesion.

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