scholarly journals RAC1 Regulates Adherens Junctions through Endocytosis of E-Cadherin

2001 ◽  
Vol 12 (4) ◽  
pp. 847-862 ◽  
Author(s):  
Nasreen Akhtar ◽  
Neil A. Hotchin
Keyword(s):  
Cell Adhesion ◽  
Fluorescent Protein ◽  
Adherens Junctions ◽  
Expression Vectors ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Cell Contacts ◽  
Green Fluorescent ◽  
E Cadherin ◽  
Cell Cell

The establishment of cadherin-dependent cell–cell contacts in human epidermal keratinocytes are known to be regulated by the Rac1 small GTP-binding protein, although the mechanisms by which Rac1 participates in the assembly or disruption of cell–cell adhesion are not well understood. In this study we utilized green fluorescent protein (GFP)-tagged Rac1 expression vectors to examine the subcellular distribution of Rac1 and its effects on E-cadherin–mediated cell–cell adhesion. Microinjection of keratinocytes with constitutively active Rac1 resulted in cell spreading and disruption of cell–cell contacts. The ability of Rac1 to disrupt cell–cell adhesion was dependent on colony size, with large established colonies being resistant to the effects of active Rac1. Disruption of cell–cell contacts in small preconfluent colonies was achieved through the selective recruitment of E-cadherin–catenin complexes to the perimeter of multiple large intracellular vesicles, which were bounded by GFP-tagged L61Rac1. Similar vesicles were observed in noninjected keratinocytes when cell–cell adhesion was disrupted by removal of extracellular calcium or with the use of an E-cadherin blocking antibody. Moreover, formation of these structures in noninjected keratinocytes was dependent on endogenous Rac1 activity. Expression of GFP-tagged effector mutants of Rac1 in keratinocytes demonstrated that reorganization of the actin cytoskeleton was important for vesicle formation. Characterization of these Rac1-induced vesicles revealed that they were endosomal in nature and tightly colocalized with the transferrin receptor, a marker for recycling endosomes. Expression of GFP-L61Rac1 inhibited uptake of transferrin-biotin, suggesting that the endocytosis of E-cadherin was a clathrin-independent mechanism. This was supported by the observation that caveolin, but not clathrin, localized around these structures. Furthermore, an inhibitory form of dynamin, known to inhibit internalization of caveolae, inhibited formation of cadherin vesicles. Our data suggest that Rac1 regulates adherens junctions via clathrin independent endocytosis of E-cadherin.

scholarly journals Mechanisms of Epithelial Cell–Cell Adhesion and Cell Compaction Revealed by High-resolution Tracking of E-Cadherin– Green Fluorescent Protein

1998 ◽  
Vol 142 (4) ◽  
pp. 1105-1119 ◽  
Author(s):  
Cynthia L. Adams ◽  
Yih-Tai Chen ◽  
Stephen J Smith ◽  
W. James Nelson
Keyword(s):  
Cell Adhesion ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Time Lapse ◽  
Cell Contact ◽  
Membrane Contacts ◽  
Green Fluorescent ◽  
Actin Cables ◽  
E Cadherin ◽  
Cell Cell

Cadherin-mediated adhesion initiates cell reorganization into tissues, but the mechanisms and dynamics of such adhesion are poorly understood. Using time-lapse imaging and photobleach recovery analyses of a fully functional E-cadherin/GFP fusion protein, we define three sequential stages in cell–cell adhesion and provide evidence for mechanisms involving E-cadherin and the actin cytoskeleton in transitions between these stages. In the first stage, membrane contacts between two cells initiate coalescence of a highly mobile, diffuse pool of cell surface E-cadherin into immobile punctate aggregates along contacting membranes. These E-cadherin aggregates are spatially coincident with membrane attachment sites for actin filaments branching off from circumferential actin cables that circumscribe each cell. In the second stage, circumferential actin cables near cell–cell contact sites separate, and the resulting two ends of the cable swing outwards to the perimeter of the contact. Concomitantly, subsets of E-cadherin puncta are also swept to the margins of the contact where they coalesce into large E-cadherin plaques. This reorganization results in the formation of a circumferential actin cable that circumscribes both cells, and is embedded into each E-cadherin plaque at the contact margin. At this stage, the two cells achieve maximum contact, a process referred to as compaction. These changes in E-cadherin and actin distributions are repeated when additional single cells adhere to large groups of cells. The third stage of adhesion occurs as additional cells are added to groups of >3 cells; circumferential actin cables linked to E-cadherin plaques on adjacent cells appear to constrict in a purse-string action, resulting in the further coalescence of individual plaques into the vertices of multicell contacts. The reorganization of E-cadherin and actin results in the condensation of cells into colonies. We propose a model to explain how, through strengthening and compaction, E-cadherin and actin cables coordinate to remodel initial cell–cell contacts to the final condensation of cells into colonies.

scholarly journals A molecular mechanism directly linking E-cadherin adhesion to initiation of epithelial cell surface polarity

2007 ◽  
Vol 178 (2) ◽  
pp. 323-335 ◽  
Author(s):  
Lene N. Nejsum ◽  
W. James Nelson
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cell ◽  
Cell Surface ◽  
Basolateral Membrane ◽  
Surface Polarity ◽  
Cell Contacts ◽  
Protein Receptors ◽  
Epithelial Cell Surface ◽  
E Cadherin ◽  
Cell Cell

Mechanisms involved in maintaining plasma membrane domains in fully polarized epithelial cells are known, but when and how directed protein sorting and trafficking occur to initiate cell surface polarity are not. We tested whether establishment of the basolateral membrane domain and E-cadherin–mediated epithelial cell–cell adhesion are mechanistically linked. We show that the basolateral membrane aquaporin (AQP)-3, but not the equivalent apical membrane AQP5, is delivered in post-Golgi structures directly to forming cell–cell contacts where it co-accumulates precisely with E-cadherin. Functional disruption of individual components of a putative lateral targeting patch (e.g., microtubules, the exocyst, and soluble N-ethylmaleimide–sensitive factor attachment protein receptors) did not inhibit cell–cell adhesion or colocalization of the other components with E-cadherin, but each blocked AQP3 delivery to forming cell–cell contacts. Thus, components of the lateral targeting patch localize independently of each other to cell–cell contacts but collectively function as a holocomplex to specify basolateral vesicle delivery to nascent cell–cell contacts and immediately initiate cell surface polarity.

scholarly journals Role of Nectin in Formation of E-Cadherin–based Adherens Junctions in Keratinocytes: Analysis with the N-Cadherin Dominant Negative Mutant

2003 ◽  
Vol 14 (4) ◽  
pp. 1597-1609 ◽  
Author(s):  
Yoshinari Tanaka ◽  
Hiroyuki Nakanishi ◽  
Shigeki Kakunaga ◽  
Noriko Okabe ◽  
Tomomi Kawakatsu ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Adherens Junctions ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Adhesion Activity ◽  
Negative Mutant ◽  
E Cadherin ◽  
Cell Cell

E-Cadherin is a Ca2+-dependent cell-cell adhesion molecule at adherens junctions (AJs) of epithelial cells. A fragment of N-cadherin lacking its extracellular region serves as a dominant negative mutant (DN) and inhibits cell-cell adhesion activity of E-cadherin, but its mode of action remains to be elucidated. Nectin is a Ca2+-independent immunoglobulin-like cell-cell adhesion molecule at AJs and is associated with E-cadherin through their respective peripheral membrane proteins, afadin and catenins, which connect nectin and cadherin to the actin cytoskeleton, respectively. We showed here that overexpression of nectin capable of binding afadin, but not a mutant incapable of binding afadin, reduced the inhibitory effect of N-cadherin DN on the cell-cell adhesion activity of E-cadherin in keratinocytes. Overexpressed nectin recruited N-cadherin DN to the nectin-based cell-cell adhesion sites in an afadin-dependent manner. Moreover, overexpression of nectin enhanced the E-cadherin–based cell-cell adhesion activity. These results suggest that N-cadherin DN competitively inhibits the association of the endogenous nectin-afadin system with the endogenous E-cadherin-catenin system and thereby reduces the cell-cell adhesion activity of E-cadherin. Thus, nectin plays a role in the formation of E-cadherin–based AJs in keratinocytes.

Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes

1996 ◽  
Vol 109 (13) ◽  
pp. 3013-3023 ◽  
Author(s):  
A.J. Zhu ◽  
F.M. Watt
Keyword(s):  
Cell Adhesion ◽  
Terminal Differentiation ◽  
Intercellular Adhesion ◽  
Dominant Negative ◽  
Epidermal Keratinocytes ◽  
Dominant Negative Mutant ◽  
Beta Catenin ◽  
Human Epidermal Keratinocytes ◽  
Negative Mutant ◽  
E Cadherin

Cell adhesion molecules are not only required for maintenance of tissue integrity, but also regulate many aspects of cell behaviour, including growth and differentiation. While the regulatory functions of integrin extracellular matrix receptors in keratinocytes are well established, such functions have not been investigated for the primary receptors that mediate keratinocyte intercellular adhesion, the cadherins. To examine cadherin function in normal human epidermal keratinocytes we used a retroviral vector to introduce a dominant negative E-cadherin mutant, consisting of the extracellular domain of H-2Kd and the transmembrane and cytoplasmic domains of E-cadherin. As a control a vector containing the same construct, but with the catenin binding site destroyed, was prepared. High levels of expression of the constructs were achieved; the dominant negative mutant, but not the control, formed complexes with alpha-, beta- and gamma-catenin. In cells expressing the dominant negative mutant there was a 5-fold decrease in the level of endogenous cadherins and a 3-fold increase in the level of beta-catenin. Cell-cell adhesion and stratification were inhibited by the dominant negative mutant and desmosome formation was reduced. Expression of the mutant resulted in reduced levels of the alpha 2 beta 1 and alpha 3 beta 1 integrins and increased cell motility, providing further evidence for cross-talk between cadherins and the beta 1 integrins. In view of the widely documented loss of E-cadherin in keratinocyte tumours it was surprising that the dominant negative mutant had an inhibitory effect on keratinocyte proliferation and stimulated terminal differentiation even under conditions in which intercellular adhesion was prevented. These results establish a role for cadherins in regulating keratinocyte growth and differentiation and raise interesting questions as to the relative importance of cell adhesion-dependent and -independent mechanisms.

Involvement of a Rab8-like protein of Dictyostelium discoideum, Sas1, in the formation of membrane extensions, secretion and adhesion during development

Microbiology ◽  
2004 ◽  
Vol 150 (8) ◽  
pp. 2513-2525 ◽  
Author(s):  
Rhonda R. Powell ◽  
Lesly A. Temesvari
Keyword(s):  
Cell Adhesion ◽  
Dictyostelium Discoideum ◽  
Fluorescent Protein ◽  
Aggregate Size ◽  
Developmental Context ◽  
Cellular Events ◽  
Membrane Protrusions ◽  
A Cell ◽  
Green Fluorescent ◽  
Cell Cell

Establishment of cell–cell adhesions, regulation of actin, and secretion are critical during development. Rab8-like GTPases have been shown to modulate these cellular events, suggesting an involvement in developmental processes. To further elucidate the function of Rab8-like GTPases in a developmental context, a Rab8-related protein (Sas1) of Dictyostelium discoideum was examined, the expression of which increases at the onset of development. Dictyostelium cell lines expressing inactive (N128I mutant) and constitutively active (Q74L mutant) Sas1 as green fluorescent protein (GFP)-Sas1 chimeras were generated. Cells expressing Sas1Q74L displayed numerous actin-rich membrane protrusions, increased secretion, and were unable to complete development. In particular, these cells demonstrated a reduction in adhesion as well as in the levels of a cell adhesion molecule, gp24 (DdCAD-1). In contrast, cells expressing Sas1N128I exhibited increased cell–cell adhesion and increased levels of gp24. Counting factor is a multisubunit signalling complex that is secreted in early development and controls aggregate size by negatively regulating the levels of cell adhesion molecules, including gp24. Interestingly, the Sas1Q74L mutant demonstrated increased levels of extracellular countin, a subunit of counting factor, suggesting that Sas1 may regulate trafficking of counting factor components. Together, the data suggest that Sas1 may be a key regulator of actin, adhesion and secretion during development.

scholarly journals Microtubule Disruption in Keratinocytes Induces Cell-Cell Adhesion through Activation of Endogenous E-Cadherin

2001 ◽  
Vol 12 (7) ◽  
pp. 1983-1993 ◽  
Author(s):  
Sun-Ho Kee ◽  
Peter M. Steinert
Keyword(s):  
Cell Adhesion ◽  
Cell Lines ◽  
Epidermal Keratinocytes ◽  
Cell Periphery ◽  
Microtubule Disruption ◽  
Low Calcium ◽  
High Calcium ◽  
Long Term Treatment ◽  
E Cadherin ◽  
Cell Cell

The association of the cytoskeleton with the cadherin–catenin complex is essential for strong cell-cell adhesion in epithelial cells. In this study, we have investigated the effect of microtubule organization on cell-cell adhesion in differentiating keratinocytes. When microtubules of normal human epidermal keratinocytes (NHEKs) grown in low calcium media (0.05 mM) were disrupted with nocodazole or colcemid, cell-cell adhesion was induced through relocalization of the E-cadherin–catenin–actin complex to the cell periphery. This was accompanied by actin polymerization. Also, it was found that microtubule disruption-induced cell-cell adhesion was significantly reduced in more advanced differentiated keratinocytes. For example, when NHEK cells cultured under high calcium (1.2 mM) for 8 d and then in low calcium for 1 d were treated with nocodazole, there was no induction of cell-cell adhesion. Also long-term treatment of a phorbol ester for 48 h inhibited nocodazole-induced cell-cell adhesion of NHEK. Furthermore, this nocodazole-induced cell-cell adhesion could be observed in squamous cancer cell lines (A431 and SCC-5, -9, and -25) under low calcium condition, but not in the keratinocyte cell lines derived from normal epidermis (HaCaT, RHEK). On the other hand, HaCaT cells continuously cultivated in low calcium media regained a less differentiated phenotype such as decreased expression of cytokeratin 10, and increased K5; these changes were accompanied with inducibility of cell-cell adhesion by nocodazole. Together, our results suggest that microtubule disruption can induce the cell-cell adhesion via activation of endogenous E-cadherin in non- or early differentiating keratinocytes. However, this is no longer possible in advanced terminally differentiating keratinocytes, possibly due to irreversible changes effected by cell envelope barrier formation.

scholarly journals Inhibition of N-glycosylation by tunicamycin attenuates cell–cell adhesion via impaired desmosome formation in normal human epidermal keratinocytes

2018 ◽  
Vol 38 (6) ◽  
Author(s):  
Seon-Pil Jin ◽  
Jin Ho Chung
Keyword(s):  
Cell Adhesion ◽  
Adhesive Strength ◽  
Epidermal Keratinocytes ◽  
Primary Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Tissue Integrity ◽  
Cell Adhesive ◽  
Protein Functions ◽  
The Stability ◽  
Cell Cell

N-Glycosylation affects protein functions such as location, stability, and susceptibility to proteases. Desmosomes in keratinocytes are essential to maintain epidermal tissue integrity to protect against environmental insults. However, it is not yet known whether N-glycosylation affects desmosomal functions in primary keratinocytes. Tunicamycin is an inhibitor of N-glycosylation that has been a useful tool in glycobiology. Therefore, we investigated the effect of inhibiting N-glycosylation by tunicamycin treatment on desmosomes in primary keratinocytes. In our experiments, cell–cell adhesive strength was reduced in tunicamycin-treated primary keratinocytes. TEM showed that desmosome formation was impaired by tunicamycin. Desmogleins (Dsgs) 1 and 3, which constitute the core structure of desmosomes, were well transported to the cell–cell borders, but the amount decreased and showed an aberrant distribution at the cell borders in tunicamycin-treated keratinocytes. The stability of both desmoglein proteins was also reduced, and they were degraded through both proteasomal and lysosomal pathways, although inhibiting degradation did not restore the cell–cell adhesion. Finally, tunicamycin induced desmosomal instability, enhancing their disassembly. In conclusion, these results indicate that N-glycosylation is critical to the desmosome complex to maintain cell–cell adhesive strength in primary keratinocytes.

scholarly journals Different effects of dominant negative mutants of desmocollin and desmoglein on the cell-cell adhesion of keratinocytes

2000 ◽  
Vol 113 (10) ◽  
pp. 1803-1811
Author(s):  
Y. Hanakawa ◽  
M. Amagai ◽  
Y. Shirakata ◽  
K. Sayama ◽  
K. Hashimoto
Keyword(s):  
Cell Adhesion ◽  
Adherens Junctions ◽  
Dominant Negative ◽  
Cell Contact ◽  
Beta Catenin ◽  
Hacat Cells ◽  
Subsequent Formation ◽  
Contact Sites ◽  
E Cadherin ◽  
Cell Cell

Desmosomes contain two types of cadherin: desmocollin (Dsc) and desmoglein (Dsg). In this study, we examined the different roles that Dsc and Dsg play in the formation of desmosomes, by using dominant-negative mutants. We constructed recombinant adenoviruses (Ad) containing truncated mutants of E-cadherin, desmocollin 3a, and desmoglein 3 lacking a large part of their extracellular domains (EcaddeltaEC, Dsc3adeltaEC, Dsg3deltaEC), using the Cre-loxP Ad system to circumvent the problem of the toxicity of the mutants to virus-producing cells. When Dsc3adeltaEC Ad-infected HaCaT cells were cultured with high levels of calcium, E-cadherin and beta-catenin, which are marker molecules for the adherens junction, disappeared from the cell-cell contact sites, and cell-cell adhesion was disrupted. This also occurred in the cells infected with EcaddeltaEC Ad. With Dsg3deltaEC Ad infection, keratin insertion at the cell-cell contact sites was inhibited and desmoplakin, a marker of desmosomes, was stained in perinuclear dots while the adherens junctions remained intact. Dsc3adeltaEC Ad inhibited the induction of adherens junctions and the subsequent formation of desmosomes with the calcium shift, while Dsg3deltaEC Ad only inhibited the formation of desmosomes. To further determine whether Dsc3adeltaEC directly affected adherens junctions, mouse fibroblast L cells transfected with E-cadherin (LEC5) were infected with these mutant Ads. Both Dsc3adeltaEC and EcaddeltaEC inhibited the cell-cell adhesion of LEC5 cells, as determined by the cell aggregation assay, while Dsg3deltaEC did not. These results indicate that the dominant negative effects of Dsg3deltaEC were restricted to desmosomes, while those of Dsc3adeltaEC were observed in both desmosomes and adherens junctions. Furthermore, the cytoplasmic domain of Dsc3adeltaEC coprecipitated both plakoglobin and beta-catenin in HaCaT cells. In addition, beta-catenin was found to bind the endogenous Dsc in HaCaT cells. These findings lead us to speculate that Dsc interacts with components of the adherens junctions through beta-catenin, and plays a role in nucleating desmosomes after the adherens junctions have been established.

scholarly journals Early Events in Actin Cytoskeleton Dynamics and E-Cadherin-Mediated Cell-Cell Adhesion during Epithelial-Mesenchymal Transition

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 578 ◽  
Author(s):  
Irina Y. Zhitnyak ◽  
Svetlana N. Rubtsova ◽  
Nikita I. Litovka ◽  
Natalya A. Gloushankova
Keyword(s):  
Cell Adhesion ◽  
Epithelial Mesenchymal Transition ◽  
Immunofluorescent Staining ◽  
Actin Binding ◽  
Mesenchymal Transition ◽  
Actin Bundle ◽  
Cell Contacts ◽  
The Stability ◽  
E Cadherin ◽  
Cell Cell

Epithelial-mesenchymal transition (EMT) plays an important role in development and also in initiation of metastasis during cancer. Disruption of cell-cell contacts during EMT allowing cells to detach from and migrate away from their neighbors remains poorly understood. Using immunofluorescent staining and live-cell imaging, we analyzed early events during EMT induced by epidermal growth factor (EGF) in IAR-20 normal epithelial cells. Control cells demonstrated stable adherens junctions (AJs) and robust contact paralysis, whereas addition of EGF caused rapid dynamic changes at the cell-cell boundaries: fragmentation of the circumferential actin bundle, assembly of actin network in lamellipodia, and retrograde flow. Simultaneously, an actin-binding protein EPLIN was phosphorylated, which may have decreased the stability of the circumferential actin bundle. Addition of EGF caused gradual replacement of linear E-cadherin–based AJs with dynamic and unstable punctate AJs, which, unlike linear AJs, colocalized with the mechanosensitive protein zyxin, confirming generation of centripetal force at the sites of cell-cell contacts during EMT. Our data show that early EMT promotes heightened dynamics at the cell-cell boundaries—replacement of stable AJs and actin structures with dynamic ones—which results in overall weakening of cell-cell adhesion, thus priming the cells for front-rear polarization and eventual migration.

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