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Force measurements in E-cadherin-mediated cell doublets reveal rapid adhesion strengthened by actin cytoskeleton remodeling through Rac and Cdc42.

Chu YS, Thomas WA, Eder O, Pincet F, Perez E, Thiery JP, Dufour S - J. Cell Biol. (2004)

Bottom Line: Separation force depended on the homophilic interaction of functional cadherins at the cell surface, increasing with the duration of contact and with cadherin levels.Severing the link between cadherin and the actin cytoskeleton or disrupting actin polymerization did not affect initiation of cadherin-mediated adhesion, but prevented it from developing and becoming stronger over time.Rac and Cdc42, the Rho-like small GTPases, were activated when E-cadherin-expressing cells formed aggregates in suspension.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique-Institut Curie, Paris, France.

ABSTRACT
We have used a modified, dual pipette assay to quantify the strength of cadherin-dependent cell-cell adhesion. The force required to separate E-cadherin-expressing paired cells in suspension was measured as an index of intercellular adhesion. Separation force depended on the homophilic interaction of functional cadherins at the cell surface, increasing with the duration of contact and with cadherin levels. Severing the link between cadherin and the actin cytoskeleton or disrupting actin polymerization did not affect initiation of cadherin-mediated adhesion, but prevented it from developing and becoming stronger over time. Rac and Cdc42, the Rho-like small GTPases, were activated when E-cadherin-expressing cells formed aggregates in suspension. Overproduction of the dominant negative form of Rac or Cdc42 permitted initial E-cadherin-based adhesion but affected its later development; the dominant active forms prevented cell adhesion outright. Our findings highlight the crucial roles played by Rac, Cdc42, and actin cytoskeleton dynamics in the development and regulation of strong cell adhesion, defined in terms of mechanical forces.

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The time-dependent increase in SF depends on the connection of cadherin to the actin cytoskeleton. (A) Schematic representation of the structure of wild-type cadherin, E-cadherin lacking the cytoplasmic domain (Ecad-Δcyto), and E-cadherin–α-cat chimera (EαMC) expressed by transiently transfected S180 cells. FACS analysis of transiently cotransfected cells expressing Ecad (B), Ecad-Δcyto (B), EαMC (C), or E58 cells (C) with anti–β-cat (B and C, top, white peaks), anti–E-cadherin ECCD2 antibody (B and C, bottom, white peaks), or control antibodies (black peaks). (D and E) Mean SF for 30-s, 4- and 30-min doublets of GFP-positive cells expressing E-cadherin (D, black bars), Ecad-Δcyto (D, white bars), EαMC chimera (E, gray bars), and doublets of E58 cells (E, black bars). Immunodetection of Ecad-Δcyto (F) and EαMC (G) proteins in representative doublets formed after 30-min aggregation in suspension. Bar, 10 μm.
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fig5: The time-dependent increase in SF depends on the connection of cadherin to the actin cytoskeleton. (A) Schematic representation of the structure of wild-type cadherin, E-cadherin lacking the cytoplasmic domain (Ecad-Δcyto), and E-cadherin–α-cat chimera (EαMC) expressed by transiently transfected S180 cells. FACS analysis of transiently cotransfected cells expressing Ecad (B), Ecad-Δcyto (B), EαMC (C), or E58 cells (C) with anti–β-cat (B and C, top, white peaks), anti–E-cadherin ECCD2 antibody (B and C, bottom, white peaks), or control antibodies (black peaks). (D and E) Mean SF for 30-s, 4- and 30-min doublets of GFP-positive cells expressing E-cadherin (D, black bars), Ecad-Δcyto (D, white bars), EαMC chimera (E, gray bars), and doublets of E58 cells (E, black bars). Immunodetection of Ecad-Δcyto (F) and EαMC (G) proteins in representative doublets formed after 30-min aggregation in suspension. Bar, 10 μm.

Mentions: We determined the role of the E-cadherin cytoplasmic domain and its partners in the establishment of cell adhesion by comparing SF in cells expressing wild-type and cytoplasmically modified E-cadherins. Parental S180 cells were transiently cotransfected with pEGFPC1 and a plasmid encoding E-cadherin, E-cadherin lacking the cytoplasmic domain (Ecad-Δcyto) or E-cadherin–α-cat chimera (EαMC; Ozawa, 2002; Fig. 5 A). FACS analysis revealed that GFP-producing cells expressed higher levels of E-cadherin or Ecad-Δcyto (Fig. 5 B, bottom) than EαMC (Fig. 5 C, bottom), whereas the GFP-positive EαMC transfectants expressed an amount of mutant E-cadherin similar to that of the E-cadherin in the expressor clone E58 (Fig. 5 C, bottom). Only cells expressing E-cadherin were shown to coexpress β-cat (Fig. 5, B and C, top), indicating that the lack of a β-cat binding site prevented the mutated cadherins from recruiting this cytoplasmic partner.


Force measurements in E-cadherin-mediated cell doublets reveal rapid adhesion strengthened by actin cytoskeleton remodeling through Rac and Cdc42.

Chu YS, Thomas WA, Eder O, Pincet F, Perez E, Thiery JP, Dufour S - J. Cell Biol. (2004)

The time-dependent increase in SF depends on the connection of cadherin to the actin cytoskeleton. (A) Schematic representation of the structure of wild-type cadherin, E-cadherin lacking the cytoplasmic domain (Ecad-Δcyto), and E-cadherin–α-cat chimera (EαMC) expressed by transiently transfected S180 cells. FACS analysis of transiently cotransfected cells expressing Ecad (B), Ecad-Δcyto (B), EαMC (C), or E58 cells (C) with anti–β-cat (B and C, top, white peaks), anti–E-cadherin ECCD2 antibody (B and C, bottom, white peaks), or control antibodies (black peaks). (D and E) Mean SF for 30-s, 4- and 30-min doublets of GFP-positive cells expressing E-cadherin (D, black bars), Ecad-Δcyto (D, white bars), EαMC chimera (E, gray bars), and doublets of E58 cells (E, black bars). Immunodetection of Ecad-Δcyto (F) and EαMC (G) proteins in representative doublets formed after 30-min aggregation in suspension. Bar, 10 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172605&req=5

fig5: The time-dependent increase in SF depends on the connection of cadherin to the actin cytoskeleton. (A) Schematic representation of the structure of wild-type cadherin, E-cadherin lacking the cytoplasmic domain (Ecad-Δcyto), and E-cadherin–α-cat chimera (EαMC) expressed by transiently transfected S180 cells. FACS analysis of transiently cotransfected cells expressing Ecad (B), Ecad-Δcyto (B), EαMC (C), or E58 cells (C) with anti–β-cat (B and C, top, white peaks), anti–E-cadherin ECCD2 antibody (B and C, bottom, white peaks), or control antibodies (black peaks). (D and E) Mean SF for 30-s, 4- and 30-min doublets of GFP-positive cells expressing E-cadherin (D, black bars), Ecad-Δcyto (D, white bars), EαMC chimera (E, gray bars), and doublets of E58 cells (E, black bars). Immunodetection of Ecad-Δcyto (F) and EαMC (G) proteins in representative doublets formed after 30-min aggregation in suspension. Bar, 10 μm.
Mentions: We determined the role of the E-cadherin cytoplasmic domain and its partners in the establishment of cell adhesion by comparing SF in cells expressing wild-type and cytoplasmically modified E-cadherins. Parental S180 cells were transiently cotransfected with pEGFPC1 and a plasmid encoding E-cadherin, E-cadherin lacking the cytoplasmic domain (Ecad-Δcyto) or E-cadherin–α-cat chimera (EαMC; Ozawa, 2002; Fig. 5 A). FACS analysis revealed that GFP-producing cells expressed higher levels of E-cadherin or Ecad-Δcyto (Fig. 5 B, bottom) than EαMC (Fig. 5 C, bottom), whereas the GFP-positive EαMC transfectants expressed an amount of mutant E-cadherin similar to that of the E-cadherin in the expressor clone E58 (Fig. 5 C, bottom). Only cells expressing E-cadherin were shown to coexpress β-cat (Fig. 5, B and C, top), indicating that the lack of a β-cat binding site prevented the mutated cadherins from recruiting this cytoplasmic partner.

Bottom Line: Separation force depended on the homophilic interaction of functional cadherins at the cell surface, increasing with the duration of contact and with cadherin levels.Severing the link between cadherin and the actin cytoskeleton or disrupting actin polymerization did not affect initiation of cadherin-mediated adhesion, but prevented it from developing and becoming stronger over time.Rac and Cdc42, the Rho-like small GTPases, were activated when E-cadherin-expressing cells formed aggregates in suspension.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique-Institut Curie, Paris, France.

ABSTRACT
We have used a modified, dual pipette assay to quantify the strength of cadherin-dependent cell-cell adhesion. The force required to separate E-cadherin-expressing paired cells in suspension was measured as an index of intercellular adhesion. Separation force depended on the homophilic interaction of functional cadherins at the cell surface, increasing with the duration of contact and with cadherin levels. Severing the link between cadherin and the actin cytoskeleton or disrupting actin polymerization did not affect initiation of cadherin-mediated adhesion, but prevented it from developing and becoming stronger over time. Rac and Cdc42, the Rho-like small GTPases, were activated when E-cadherin-expressing cells formed aggregates in suspension. Overproduction of the dominant negative form of Rac or Cdc42 permitted initial E-cadherin-based adhesion but affected its later development; the dominant active forms prevented cell adhesion outright. Our findings highlight the crucial roles played by Rac, Cdc42, and actin cytoskeleton dynamics in the development and regulation of strong cell adhesion, defined in terms of mechanical forces.

Show MeSH
Related in: MedlinePlus