scholarly journals Cytoskeleton assembly at endothelial cell–cell contacts is regulated by αII-spectrin–VASP complexes

2008 ◽  
Vol 180 (1) ◽  
pp. 205-219 ◽  
Author(s):  
Peter M. Benz ◽  
Constanze Blume ◽  
Jan Moebius ◽  
Chris Oschatz ◽  
Kai Schuh ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Ectopic Expression ◽  
Sh3 Domain ◽  
Fiber Formation ◽  
Cell Junctions ◽  
Cortical Actin ◽  
Differential Proteomics ◽  
Cell Contacts ◽  
Actin Fiber ◽  
Cell Cell

Directed cortical actin assembly is the driving force for intercellular adhesion. Regulated by phosphorylation, vasodilator-stimulated phosphoprotein (VASP) participates in actin fiber formation. We screened for endothelial proteins, which bind to VASP, dependent on its phosphorylation status. Differential proteomics identified αII-spectrin as such a VASP-interacting protein. αII-Spectrin binds to the VASP triple GP5-motif via its SH3 domain. cAMP-dependent protein kinase–mediated VASP phosphorylation at Ser157 inhibits αII-spectrin–VASP binding. VASP is dephosphorylated upon formation of cell–cell contacts and in confluent, but not in sparse cells, αII-spectrin colocalizes with nonphosphorylated VASP at cell–cell junctions. Ectopic expression of the αII-spectrin SH3 domain at cell–cell contacts translocates VASP, initiates cortical actin cytoskeleton formation, stabilizes cell–cell contacts, and decreases endothelial permeability. Conversely, the permeability of VASP-deficient endothelial cells (ECs) and microvessels of VASP-null mice increases. Reconstitution of VASP-deficient ECs rescues barrier function, whereas αII-spectrin binding-deficient VASP mutants fail to restore elevated permeability. We propose that αII-spectrin–VASP complexes regulate cortical actin cytoskeleton assembly with implications for vascular permeability.

scholarly journals Dual roles of myocardin-related transcription factors in epithelial–mesenchymal transition via slug induction and actin remodeling

2007 ◽  
Vol 179 (5) ◽  
pp. 1027-1042 ◽  
Author(s):  
Tsuyoshi Morita ◽  
Taira Mayanagi ◽  
Kenji Sobue
Keyword(s):  
Transcription Factors ◽  
Actin Cytoskeleton ◽  
Nuclear Translocation ◽  
Epithelial Mesenchymal Transition ◽  
Ectopic Expression ◽  
Cytoskeletal Proteins ◽  
Mesenchymal Transition ◽  
Cell Contacts ◽  
Tgf Β1 ◽  
Cell Cell

Epithelial–mesenchymal transition (EMT) is a critical process occurring during embryonic development and in fibrosis and tumor progression. Dissociation of cell–cell contacts and remodeling of the actin cytoskeleton are major events of the EMT. Here, we show that myocardin-related transcription factors (MRTFs; also known as MAL and MKL) are critical mediators of transforming growth factor β (TGF-β) 1–induced EMT. In all epithelial cell lines examined here, TGF-β1 triggers the nuclear translocation of MRTFs. Ectopic expression of constitutive-active MRTF-A induces EMT, whereas dominant-negative MRTF-A or knockdown of MRTF-A and -B prevents the TGF-β1–induced EMT. MRTFs form complexes with Smad3. Via Smad3, the MRTF–Smad3 complexes bind to a newly identified cis-element GCCG-like motif in the promoter region of Canis familiaris and the human slug gene, which activates slug transcription and thereby dissociation of cell–cell contacts. MRTFs also increase the expression levels of actin cytoskeletal proteins via serum response factor, thereby triggering reorganization of the actin cytoskeleton. Thus, MRTFs are important mediators of TGF-β1–induced EMT.

scholarly journals Two Sides of the Coin: Ezrin/Radixin/Moesin and Merlin Control Membrane Structure and Contact Inhibition

2019 ◽  
Vol 20 (8) ◽  
pp. 1996 ◽  
Author(s):  
Katharine A. Michie ◽  
Adam Bermeister ◽  
Neil O. Robertson ◽  
Sophia C. Goodchild ◽  
Paul M. G. Curmi
Keyword(s):  
Actin Cytoskeleton ◽  
Protein Complexes ◽  
Membrane Vesicle ◽  
Contact Inhibition ◽  
Cell Junctions ◽  
Cellular Membranes ◽  
Membrane Structures ◽  
Cortical Actin ◽  
Erm Proteins ◽  
Structural Conservation

The merlin-ERM (ezrin, radixin, moesin) family of proteins plays a central role in linking the cellular membranes to the cortical actin cytoskeleton. Merlin regulates contact inhibition and is an integral part of cell–cell junctions, while ERM proteins, ezrin, radixin and moesin, assist in the formation and maintenance of specialized plasma membrane structures and membrane vesicle structures. These two protein families share a common evolutionary history, having arisen and separated via gene duplication near the origin of metazoa. During approximately 0.5 billion years of evolution, the merlin and ERM family proteins have maintained both sequence and structural conservation to an extraordinary level. Comparing crystal structures of merlin-ERM proteins and their complexes, a picture emerges of the merlin-ERM proteins acting as switchable interaction hubs, assembling protein complexes on cellular membranes and linking them to the actin cytoskeleton. Given the high level of structural conservation between the merlin and ERM family proteins we speculate that they may function together.

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 Influence of aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 on epithelial differentiation and organization during lung development

2020 ◽  
Vol 319 (2) ◽  
pp. L369-L379
Author(s):  
Daniel D. Lee ◽  
Alexandra Hochstetler ◽  
Eric Sah ◽  
Haiming Xu ◽  
Chinn-Woan Lowe ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Epithelial Cell ◽  
Actin Cytoskeleton ◽  
Cross Talk ◽  
Lung Development ◽  
Trna Synthetase ◽  
Cell Junctions ◽  
Aminoacyl Trna Synthetase ◽  
Multifunctional Protein ◽  
Cell Cell

Proper development of the respiratory bronchiole and alveolar epithelium proceeds through coordinated cross talk between the interface of epithelium and neighboring mesenchyme. Signals that facilitate and coordinate the cross talk as the bronchial forming canalicular stage transitions to construction of air-exchanging capillary-alveoli niche in the alveolar stage are poorly understood. Expressed within this decisive region, levels of aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1) inversely correlate with the maturation of the lung. The present study addresses the role of AIMP1 in lung development through the generation and characterization of Aimp1−/− mutant mice. Mating of Aimp1+/− produced offspring in expected Mendelian ratios throughout embryonic development. However, newborn Aimp1−/− pups exhibited neonatal lethality with mild cyanosis. Imaging both structure and ultrastructure of Aimp1−/− lungs showed disorganized bronchial epithelium, decreased type I but not type II cell differentiation, increased distal vessels, and disruption of E-cadherin deposition in cell-cell junctions. Supporting the in vivo findings of disrupted epithelial cell-cell junctions, in vitro biochemical experiments show that a portion of AIMP1 binds to phosphoinositides, the lipid anchor of proteins that have a fundamental role in both cellular membrane and actin cytoskeleton organization; a dramatic disruption in F-actin cytoskeleton was observed in Aimp1−/− mouse embryonic fibroblasts. Such observed structural defects may lead to disrupted cell-cell boundaries. Together, these results suggest a requirement of AIMP1 in epithelial cell differentiation in proper lung development.

scholarly journals N-Cadherin Association with Lipid Rafts Regulates Its Dynamic Assembly at Cell-Cell Junctions in C2C12 Myoblasts

2005 ◽  
Vol 16 (5) ◽  
pp. 2168-2180 ◽  
Author(s):  
Marie Causeret ◽  
Nicolas Taulet ◽  
Franck Comunale ◽  
Cyril Favard ◽  
Cécile Gauthier-Rouvière
Keyword(s):  
Plasma Membrane ◽  
Cell Adhesion ◽  
Lipid Rafts ◽  
Membrane Lipid ◽  
Cell Junctions ◽  
Cell Contact ◽  
C2c12 Myoblasts ◽  
Cell Contacts ◽  
Dynamic Assembly ◽  
Cell Cell

Cadherins are homophilic cell-cell adhesion molecules implicated in cell growth, differentiation, and organization into tissues during embryonic development. They accumulate at cell-cell contact sites and act as adhesion-activated signaling receptors. Here, we show that the dynamic assembly of N-cadherin at cell-cell contacts involves lipid rafts. In C2C12 myoblasts, immunofluorescence and biochemical experiments demonstrate that N-cadherin present at cell-cell contacts is colocalized with lipid rafts. Disruption of lipid rafts leads to the inhibition of cell-cell adhesion and disorganization of N-cadherin–dependent cell-cell contacts without modifying the association of N-cadherin with catenins and its availability at the plasma membrane. Fluorescent recovery after photobleaching experiments demonstrate that at the dorsal plasma membrane, lipid rafts are not directly involved in the diffusional mobility of N-cadherin. In contrast, at cell-cell junctions N-cadherin association with lipid rafts allows its stabilization enabling the formation of a functional adhesive complex. We show that lipid rafts, as homophilic interaction and F-actin association, stabilize cadherin-dependent adhesive complexes. Homophilic interactions and F-actin association of N-cadherin are both required for its association to lipid rafts. We thus identify lipid rafts as new regulators of cadherin-mediated cell adhesion.

The Basic Helix-Loop-Helix TAL1/SCL and Its Partners E47 and LMO2 Act Upstream of the VE-Cadherin Gene in Endothelial Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1795-1795
Author(s):  
Virginie Deleuze ◽  
Elias Chalhoub ◽  
Rawan El-Hajj ◽  
Christiane Dohet ◽  
Mikael Le Clech ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Ectopic Expression ◽  
Cell Junctions ◽  
Small Interfering Rnas ◽  
Hek 293 ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Cell Cell

Abstract The basic helix-loop-helix protein TAL-1/SCL, essential for the formation of the hematopoietic system, is also required for vascular development and more particularly for embryonic angiogenesis. We previously reported that TAL-1 acts as a positive factor for post-natal angiogenesis by stimulating endothelial morphogenesis. To understand how TAL-1 modulates angiogenesis, we investigated the functional consequences of TAL-1 silencing, mediated by small-interfering RNAs, in human primary endothelial cells (ECs). We found that TAL-1 knockdown impaired in vitro EC tubulomorphogenesis (in 2-D on Matrigel or 3-D in collagen I gel), with the notable absence of cell-cell contacts, a prerequisite for morphogenesis initiation. This cellular deficiency was associated with a dramatic reduction in the vascular-endothelial (VE)-cadherin at intercellular junctions, the major component of endothelial adherens junctions. In contrast, PECAM (or CD31) was present at cell-cell junctions at the same levels as control cells. Importantly, silencing of two known TAL-1-partners in hematopoietic cells, E47 or LMO2, produce the same effects as TAL-1. Accordingly, silencing of TAL-1, as well as E47 and LMO2, provoked down-regulation of VE-cadherin at both the mRNA and protein levels. Transient transfection experiments in HUVECs showed that TAL-1 and E47 regulate the VE-cadherin promoter through a specialized E-box element. Finally, endogenous VE-cadherin transcription could be directly activated in non-endothelial HEK-293 cells that neither express TAL-1 or LMO2, by the sole concomitant ectopic expression of TAL-1, E47 and LMO2. Overall, our data demonstrate that a multiprotein complex containing at least TAL-1, LMO2 and E47 act upstream of the VE-cadherin gene. We are currently performing chromatin immunoprecipitation (ChIP) to investigate whether the TAL-1-containing complex binds in vivo the VE-cadherin promoter. This study identifies VE-cadherin as an upstream TAL-1-target gene in the endothelial lineage, and provides a first clue in TAL-1 function in the control of angiogenesis.

scholarly journals Ras-GAP Controls Rho-Mediated Cytoskeletal Reorganization through Its SH3 Domain

1998 ◽  
Vol 18 (9) ◽  
pp. 5567-5578 ◽  
Author(s):  
Véronique Leblanc ◽  
Bruno Tocque ◽  
Isabelle Delumeau
Keyword(s):  
Actin Cytoskeleton ◽  
Sh3 Domain ◽  
Stress Fiber ◽  
Dependent Manner ◽  
3T3 Cells ◽  
Cortical Actin ◽  
Membrane Ruffling ◽  
Sh2 Domains ◽  
Neurite Retraction ◽  
Ras Signalling

ABSTRACT Proteins of the Ras superfamily, Ras, Rac, Rho, and Cdc42, control the remodelling of the cortical actin cytoskeleton following growth factor stimulation. A major regulator of Ras, Ras-GAP, contains several structural motifs, including an SH3 domain and two SH2 domains, and there is evidence that they harbor a signalling function. We have previously described a monoclonal antibody to the SH3 domain of Ras-GAP which blocks Ras signalling in Xenopus oocytes. We now show that microinjection of this antibody into Swiss 3T3 cells prevents the formation of actin stress fibers stimulated by growth factors or activated Ras, but not membrane ruffling. This inhibition is bypassed by coinjection of activated Rho, suggesting that the Ras-GAP SH3 domain is necessary for endogenous Rho activation. In agreement, the antibody blocks lysophosphatidic acid-induced neurite retraction in differentiated PC12 cells. Furthermore, we demonstrate that microinjection of full-length Ras-GAP triggers stress fiber polymerization in fibroblasts in an SH3-dependent manner, strongly suggesting an effector function besides its role as a Ras downregulator. These results support the idea that Ras-GAP connects the Ras and Rho pathways and, therefore, regulates the actin cytoskeleton through a mechanism which probably does not involve p190 Rho-GAP.

scholarly journals Direct laser manipulation reveals the mechanics of cell contacts in vivo

2015 ◽  
Vol 112 (5) ◽  
pp. 1416-1421 ◽  
Author(s):  
Kapil Bambardekar ◽  
Raphaël Clément ◽  
Olivier Blanc ◽  
Claire Chardès ◽  
Pierre-François Lenne
Keyword(s):  
Cell Shape ◽  
Constitutive Law ◽  
Cell Junctions ◽  
Scale Model ◽  
Tissue Morphogenesis ◽  
Cell Contacts ◽  
Cell Shape Changes ◽  
Shape Changes ◽  
Cell Cell

Cell-generated forces produce a variety of tissue movements and tissue shape changes. The cytoskeletal elements that underlie these dynamics act at cell–cell and cell–ECM contacts to apply local forces on adhesive structures. In epithelia, force imbalance at cell contacts induces cell shape changes, such as apical constriction or polarized junction remodeling, driving tissue morphogenesis. The dynamics of these processes are well-characterized; however, the mechanical basis of cell shape changes is largely unknown because of a lack of mechanical measurements in vivo. We have developed an approach combining optical tweezers with light-sheet microscopy to probe the mechanical properties of epithelial cell junctions in the early Drosophila embryo. We show that optical trapping can efficiently deform cell–cell interfaces and measure tension at cell junctions, which is on the order of 100 pN. We show that tension at cell junctions equilibrates over a few seconds, a short timescale compared with the contractile events that drive morphogenetic movements. We also show that tension increases along cell interfaces during early tissue morphogenesis and becomes anisotropic as cells intercalate during germ-band extension. By performing pull-and-release experiments, we identify time-dependent properties of junctional mechanics consistent with a simple viscoelastic model. Integrating this constitutive law into a tissue-scale model, we predict quantitatively how local deformations propagate throughout the tissue.

Assembly of adherens junctions is required for sphingosine 1-phosphate-induced matriptase accumulation and activation at mammary epithelial cell-cell contacts

2004 ◽  
Vol 286 (5) ◽  
pp. C1159-C1169 ◽  
Author(s):  
Ruei-Jiun Hung ◽  
Ia-Wen J. Hsu ◽  
Jennifer L. Dreiling ◽  
Mon-Juan Lee ◽  
Cicely A. Williams ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Adherens Junctions ◽  
Adherens Junction ◽  
Mammary Epithelial ◽  
Cell Junctions ◽  
Cell Contacts ◽  
Cytoskeletal Rearrangement ◽  
Sphingosine 1 Phosphate ◽  
Cell Cell

Sphingosine 1-phosphate (S1P), a bioactive phospholipid, simultaneously induces actin cytoskeletal rearrangements and activation of matriptase, a membrane-associated serine protease in human mammary epithelial cells. In this study, we used a monoclonal antibody selective for activated, two-chain matriptase to examine the functional relationship between these two S1P-induced events. Ten minutes after exposure of 184 A1N4 mammary epithelial cells to S1P, matriptase was observed to accumulate at cell-cell contacts. Activated matriptase first began to appear as small spots at cell-cell contacts, and then its deposits elongated along cell-cell contacts. Concomitantly, S1P induced assembly of adherens junctions and subcortical actin belts. Matriptase localization was observed to be coincident with markers of adherens junctions at cell-cell contacts but likely not to be incorporated into the tightly bound adhesion plaque. Disruption of subcortical actin belt formation and prevention of adherens junction assembly led to prevention of accumulation and activation of the protease at cell-cell contacts. These data suggest that S1P-induced accumulation and activation of matriptase depend on the S1P-induced adherens junction assembly. Although MAb M32, directed against one of the low-density lipoprotein receptor class A domains of matriptase, blocked S1P-induced activation of the enzyme, the antibody had no effect on S1P-induced actin cytoskeletal rearrangement. Together, these data indicate that actin cytoskeletal rearrangement is necessary but not sufficient for S1P-induced activation of matriptase at cell-cell contacts. The coupling of matriptase activation to adherens junction assembly and actin cytoskeletal rearrangement may serve to ensure tight control of matriptase activity, restricted to cell-cell junctions of mammary epithelial cells.

scholarly journals 14-3-3 recruits keratin intermediate filaments to mechanically sensitive cell-cell contacts

2018 ◽  
Author(s):  
Richard A. Mariani ◽  
Shalaka Paranjpe ◽  
Radek Dobrowolski ◽  
Gregory F. Weber
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cell Junctions ◽  
Fusion Construct ◽  
Cell Contacts ◽  
Keratin 19 ◽  
Keratin Filament ◽  
Cytoskeletal Networks ◽  
Keratin Intermediate Filaments ◽  
Cell Cell

AbstractIntermediate filament cytoskeletal networks simultaneously support mechanical integrity and influence signal transduction pathways. Marked remodeling of the keratin intermediate filament network accompanies collective cellular morphogenetic movements that occur during early embryonic development in the frog Xenopus laevis. While this reorganization of keratin is initiated by force transduction on cell-cell contacts mediated by C-cadherin, the mechanism by which keratin filament reorganization occurs remains poorly understood. In this work we demonstrate that 14-3-3 proteins regulate keratin reorganization dynamics in embryonic mesendoderm cells from Xenopus gastrula. 14-3-3 co-localizes with keratin filaments near cell-cell junctions in migrating mesendoderm. Co-immunoprecipitation, mass spectrometry and bioinformatic analyses indicate Keratin 19 is a target of 14-3-3 in the whole embryo and, more specifically, mesendoderm tissue. Inhibition of 14-3-3 results in both the decreased exchange of keratin subunits into filaments and blocks keratin filament recruitment toward cell-cell contacts. Synthetically coupling 14-3-3 to Keratin 19 through a unique fusion construct conversely induces the localization of this keratin population to the region of cell-cell contacts. Taken together, these findings indicate that 14-3-3 acts on keratin intermediate filaments and is involved in their reorganization to sites of cell adhesion.

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