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 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 Molecular requirements for actin-based lamella formation in Drosophila S2 cells

2003 ◽  
Vol 162 (6) ◽  
pp. 1079-1088 ◽  
Author(s):  
Stephen L. Rogers ◽  
Ursula Wiedemann ◽  
Nico Stuurman ◽  
Ronald D. Vale
Keyword(s):  
Adaptor Protein ◽  
Actin Dynamics ◽  
S2 Cells ◽  
Capping Protein ◽  
Rac Gtpases ◽  
Drosophila S2 Cells ◽  
Small Set ◽  
End Capping ◽  
Essential Set

Cell migration occurs through the protrusion of the actin-enriched lamella. Here, we investigated the effects of RNAi depletion of ∼90 proteins implicated in actin function on lamella formation in Drosophila S2 cells. Similar to in vitro reconstitution studies of actin-based Listeria movement, we find that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein). Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator. We also show that RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability. Our results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells.

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 Enhanced Piezoelectric Fibered Extracellular Matrix to Promote Cardiomyocyte Maturation and Tissue Formation: A 3D Computational Model

Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 135
Author(s):  
Pau Urdeitx ◽  
Mohamed H. Doweidar
Keyword(s):  
Extracellular Matrix ◽  
Cell Migration ◽  
Computational Models ◽  
Cell Junctions ◽  
Tissue Formation ◽  
Cardiac Muscle Cells ◽  
Cell Processes ◽  
Electrical Stimuli ◽  
Cell Cell

Mechanical and electrical stimuli play a key role in tissue formation, guiding cell processes such as cell migration, differentiation, maturation, and apoptosis. Monitoring and controlling these stimuli on in vitro experiments is not straightforward due to the coupling of these different stimuli. In addition, active and reciprocal cell–cell and cell–extracellular matrix interactions are essential to be considered during formation of complex tissue such as myocardial tissue. In this sense, computational models can offer new perspectives and key information on the cell microenvironment. Thus, we present a new computational 3D model, based on the Finite Element Method, where a complex extracellular matrix with piezoelectric properties interacts with cardiac muscle cells during the first steps of tissue formation. This model includes collective behavior and cell processes such as cell migration, maturation, differentiation, proliferation, and apoptosis. The model has employed to study the initial stages of in vitro cardiac aggregate formation, considering cell–cell junctions, under different extracellular matrix configurations. Three different cases have been purposed to evaluate cell behavior in fibered, mechanically stimulated fibered, and mechanically stimulated piezoelectric fibered extra-cellular matrix. In this last case, the cells are guided by the coupling of mechanical and electrical stimuli. Accordingly, the obtained results show the formation of more elongated groups and enhancement in cell proliferation.

scholarly journals Villin Severing Activity Enhances Actin-based Motility In Vivo

2007 ◽  
Vol 18 (3) ◽  
pp. 827-838 ◽  
Author(s):  
Céline Revenu ◽  
Matthieu Courtois ◽  
Alphée Michelot ◽  
Cécile Sykes ◽  
Daniel Louvard ◽  
...  
Keyword(s):  
Shigella Flexneri ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Loss Of Function ◽  
Mesenchymal Transition ◽  
Capping Protein ◽  
Function Strategy ◽  
In Cells

Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

scholarly journals Ezrin is an Actin Binding Protein That Regulates Sertoli Cell and Spermatid Adhesion During Spermatogenesis

Endocrinology ◽  
2014 ◽  
Vol 155 (10) ◽  
pp. 3981-3995 ◽  
Author(s):  
N. Ece Gungor-Ordueri ◽  
Elizabeth I. Tang ◽  
Ciler Celik-Ozenci ◽  
C. Yan Cheng
Keyword(s):  
Sertoli Cell ◽  
Sertoli Cells ◽  
Adherens Junction ◽  
Actin Binding ◽  
Permeability Barrier ◽  
Tight Junction Permeability ◽  
Residual Bodies ◽  
Cell Interface

Abstract During spermatogenesis, the transport of spermatids and the release of sperms at spermiation and the remodeling of the blood-testis barrier (BTB) in the seminiferous epithelium of rat testes require rapid reorganization of the actin-based cytoskeleton. However, the mechanism(s) and the regulatory molecule(s) remain unexplored. Herein we report findings that unfold the functional significance of ezrin in the organization of the testis-specific adherens junction at the spermatid-Sertoli cell interface called apical ectoplasmic specialization (ES) in the adluminal compartment and the Sertoli cell-cell interface known as basal ES at the BTB. Ezrin is expressed at the basal ES/BTB in all stages, except from late VIII to IX, of the epithelial cycle. Its knockdown by RNA interference (RNAi) in vitro perturbs the Sertoli cell tight junction-permeability barrier via a disruption of the actin microfilaments in Sertoli cells, which in turn impeded basal ES protein (eg, N-cadherin) distribution, perturbing the BTB function. These findings were confirmed by a knockdown study in vivo. However, the expression of ezrin at the apical ES is restricted to stage VIII of the cycle and limited only between step 19 spermatids and Sertoli cells. A knockdown of ezrin in vivo by RNAi was found to impede spermatid transport, causing defects in spermiation in which spermatids were embedded deep inside the epithelium, and associated with a loss of spermatid polarity. Also, ezrin was associated with residual bodies and phagosomes, and its knockdown by RNAi in the testis also impeded the transport of residual bodies/phagosomes from the apical to the basal compartment. In summary, ezrin is involved in regulating actin microfilament organization at the ES in rat testes.

Cell confluence regulates tyrosine phosphorylation of adherens junction components in endothelial cells

1997 ◽  
Vol 110 (17) ◽  
pp. 2065-2077 ◽  
Author(s):  
M.G. Lampugnani ◽  
M. Corada ◽  
P. Andriopoulou ◽  
S. Esser ◽  
W. Risau ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Phosphorylation ◽  
Adherens Junction ◽  
Cell Junctions ◽  
Molecular Organization ◽  
Beta Catenin ◽  
Cell Contacts ◽  
Cell Type Specific ◽  
Dynamic Structures ◽  
Cell Cell

In src- and ras-transformed cells, tyrosine phosphorylation of adherens junction (AJ) components is related to impairment of cell-cell adhesion. In this paper we report that in human endothelial cells (EC), tyrosine phosphorylation of AJ can be a physiological process regulated by cell density. Immunofluorescence analysis revealed that a phosphotyrosine (P-tyr) antibody could stain cell-cell junctions only in sparse or loosely confluent EC, while the staining was markedly reduced in tightly confluent cultures. This process was reversible, since on artificial wounding of EC monolayers, the cells at the migrating front reacquired P-tyr labelling at cell contacts. In EC, the major cadherin at intercellular AJ is the cell-type-specific VE-cadherin. We therefore analyzed whether this molecule was at least in part responsible for the changes in P-tyr content at cell junctions. Tyrosine phosphorylation of VE-cadherin, beta-catenin and p120, occurred in looser AJ, i.e. in recently confluent cells, and was notably reduced in tightly confluent cultures. Changes in P-tyr content paralleled changes in the molecular organization of AJ. VE-cadherin was mostly associated with beta-catenin and p120 in loose EC monolayers, while in long-confluent cells, these two catenins were largely replaced by plakoglobin. Inhibition of P-tyr phosphatases (PTPases) by PV markedly augmented the P-tyr content of VE-cadherin, which bound p120 and beta-catenin more efficiently, but not plakoglobin. Transfection experiments in CHO cells showed that p120 could bind to a VE-cadherin cytoplasmic region different from that responsible for beta-catenin binding, and PV stabilized this association. Overall these data indicate that endothelial AJ are dynamic structures that can be affected by the state of confluence of the cells. Tyrosine phosphorylation of VE-cadherin and its association to p120 and beta-catenin characterizes early cell contacts, while the formation of mature and cytoskeleton-connected junctions is accompanied by dephosphorylation and plakoglobin association.

scholarly journals A new 82-kD barbed end-capping protein (radixin) localized in the cell-to-cell adherens junction: purification and characterization.

1989 ◽  
Vol 108 (6) ◽  
pp. 2369-2382 ◽  
Author(s):  
S Tsukita ◽  
Y Hieda ◽  
S Tsukita
Keyword(s):  
Ion Exchange ◽  
Deae Cellulose ◽  
Adherens Junction ◽  
Methyl Cellulose ◽  
Molar Ratio ◽  
Electron Microscopic ◽  
Capping Protein ◽  
Control Value ◽  
Microscopic Studies ◽  
End Capping

An 82-kD protein has been purified from the undercoat of the adherens junction isolated from the rat liver. The purification scheme includes low salt extraction followed by DEAE-cellulose ion exchange, DNase I-actin affinity, and carboxyl methyl-cellulose ion exchange chromatographies. The purified 82-kD protein was essentially free of contaminants as judged by SDS-PAGE combined with silver staining. The substoichiometric 82-kD protein largely inhibited the actin filament assembly; when the molar ratio of the 82-kD protein to G-actin was 1:1,000, the viscosity was reduced to 28% of the control value. Direct electron microscopic studies revealed that the 82-kD protein selectively inhibited monomer addition at the barbed ends of actin filaments. By use of the antibody raised against the 82-kD protein, this protein was shown by immunofluorescence microscopy to be localized at the cell-to-cell adherens junction in various types of cells. In contrast, the 82-kD protein was not concentrated at the cell-to-substrate adherens junctions (focal contacts). These findings have led us to conclude that the 82-kD protein is a barbed end-capping protein which is associated with the undercoat of the cell-to-cell adherens junction. Hence, we have tentatively designated the 82-kD protein as radixin (from the Latin word radix meaning root).

scholarly journals A mechanism of Rap1-induced stabilization of endothelial cell–cell junctions

2011 ◽  
Vol 22 (14) ◽  
pp. 2509-2519 ◽  
Author(s):  
Jian J. Liu ◽  
Rebecca A. Stockton ◽  
Alexandre R. Gingras ◽  
Ararat J. Ablooglu ◽  
Jaewon Han ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Small Gtpases ◽  
Cell Junctions ◽  
Physical Interaction ◽  
Dependent Manner ◽  
Cardiovascular Development ◽  
Cavernous Malformations ◽  
Cell Cell

Activation of Rap1 small GTPases stabilizes cell–cell junctions, and this activity requires Krev Interaction Trapped gene 1 (KRIT1). Loss of KRIT1 disrupts cardiovascular development and causes autosomal dominant familial cerebral cavernous malformations. Here we report that native KRIT1 protein binds the effector loop of Rap1A but not H-Ras in a GTP-dependent manner, establishing that it is an authentic Rap1-specific effector. By modeling the KRIT1–Rap1 interface we designed a well-folded KRIT1 mutant that exhibited a ∼40-fold-reduced affinity for Rap1A and maintained other KRIT1-binding functions. Direct binding of KRIT1 to Rap1 stabilized endothelial cell–cell junctions in vitro and was required for cardiovascular development in vivo. Mechanistically, Rap1 binding released KRIT1 from microtubules, enabling it to locate to cell–cell junctions, where it suppressed Rho kinase signaling and stabilized the junctions. These studies establish that the direct physical interaction of Rap1 with KRIT1 enables the translocation of microtubule-sequestered KRIT1 to junctions, thereby supporting junctional integrity and cardiovascular development.

scholarly journals βcap73-ARF6 Interactions Modulate Cell Shape and Motility after Injury In Vitro

2003 ◽  
Vol 14 (10) ◽  
pp. 4155-4161 ◽  
Author(s):  
Kathleen N. Riley ◽  
Angel E. Maldonado ◽  
Patrice Tellier ◽  
Crislyn D'Souza-Schorey ◽  
Ira M. Herman
Keyword(s):  
Western Blot ◽  
Molecular Mechanisms ◽  
Active Form ◽  
Leading Edge ◽  
Dominant Negative ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Capping Protein ◽  
Actin Capping Protein

To understand the role that ARF6 plays in regulating isoactin dynamics and cell motility, we transfected endothelial cells (EC) with HA-tagged ARF6: the wild-type form (WT), a constitutively-active form unable to hydrolyze GTP (Q67L), and two dominant-negative forms, which are either unable to release GDP (T27N) or fail to bind nucleotide (N122I). Motility was assessed by digital imaging microscopy before Western blot analysis, coimmunoprecipitation, or colocalization studies using ARF6, β-actin, or β-actin-binding protein-specific antibodies. EC expressing ARF6-Q67L spread and close in vitro wounds at twice the control rates. EC expressing dominant-negative ARF6 fail to develop a leading edge, are unable to ruffle their membranes (N122I), and possess arborized processes. Colocalization studies reveal that the Q67L and WT ARF6-HA are enriched at the leading edge with β-actin; but T27N and N122I ARF6-HA are localized on endosomes together with the β-actin capping protein, βcap73. Coimmunoprecipitation and Western blot analyses reveal the direct association of ARF6-HA with βcap73, defining a role for ARF6 in signaling cytoskeletal remodeling during motility. Knowledge of the role that ARF6 plays in orchestrating membrane and β-actin dynamics will help to reveal molecular mechanisms regulating actin-based motility during development and disease.

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