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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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Drugs affecting actin polymerization perturb actin cytoskeleton organization and decrease SF. Confocal analysis of Ecad doublets formed in suspension under control conditions (A), in the presence of Jasp (B) or LatB (C), and labeled for actin and E-cadherin. Merged images are shown in right panels. The images correspond to a medial transverse plane of the doublet. Dose-response curve of SF for 4-min Ecad doublets in medium containing Jasp (D) or LatB (E). (F) Mean SF for 30-s, 4- and 30-min doublets in the presence of 0.1 μM LatB (black bars), 0.1 μM Jasp (gray bars), or in control medium (white bars).
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fig6: Drugs affecting actin polymerization perturb actin cytoskeleton organization and decrease SF. Confocal analysis of Ecad doublets formed in suspension under control conditions (A), in the presence of Jasp (B) or LatB (C), and labeled for actin and E-cadherin. Merged images are shown in right panels. The images correspond to a medial transverse plane of the doublet. Dose-response curve of SF for 4-min Ecad doublets in medium containing Jasp (D) or LatB (E). (F) Mean SF for 30-s, 4- and 30-min doublets in the presence of 0.1 μM LatB (black bars), 0.1 μM Jasp (gray bars), or in control medium (white bars).

Mentions: We determined the effects of Jasp and LatB on actin by labeling Ecad doublets with an anti-actin mAb (not depicted) or phalloidin-TRITC (Fig. 6, B and C, respectively). Under control conditions, paired cells displayed a uniform distribution of surface E-cadherin and cortical actin over most of the cell with higher density colocalization of both molecules at the cell–cell interface (Fig. 6 A). Treatment with Jasp at 0.1 μM caused cortical actin and E-cadherin to redistribute in a nonuniform manner everywhere. However, at the contact zone both molecules were still noticeably colocalized. Jasp at 1 μM dramatically reduced the thickness of cortical actin, produced actin aggregates throughout the cytoplasm and eliminated the characteristic E-cadherin/actin colocalization at the cell–cell interface. Immunostaining of actin with mAb or phalloidin gave similar results and showed that, for doublets in suspension, Jasp at both 0.1 and 1 μM mainly induces a disorganization of the actin network reflecting the aggregation/polymerization activity of this drug described by Cramer (1999). LatB at 0.1 μM had no marked effect on the localization of E-cadherin and actin in paired cells in suspension (Fig. 6 C) but 1 μM LatB treatment induced the formation of large actin aggregates in the cytoplasm, E-cadherin clusters at the cell surface and higher levels of E-cadherin staining in the cytoplasm. FACS analysis demonstrated that LatB or Jasp, at concentrations up to 0.3 μM, does not affect E-cadherin expression at the cell surface (unpublished data).


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)

Drugs affecting actin polymerization perturb actin cytoskeleton organization and decrease SF. Confocal analysis of Ecad doublets formed in suspension under control conditions (A), in the presence of Jasp (B) or LatB (C), and labeled for actin and E-cadherin. Merged images are shown in right panels. The images correspond to a medial transverse plane of the doublet. Dose-response curve of SF for 4-min Ecad doublets in medium containing Jasp (D) or LatB (E). (F) Mean SF for 30-s, 4- and 30-min doublets in the presence of 0.1 μM LatB (black bars), 0.1 μM Jasp (gray bars), or in control medium (white bars).
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Related In: Results  -  Collection

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fig6: Drugs affecting actin polymerization perturb actin cytoskeleton organization and decrease SF. Confocal analysis of Ecad doublets formed in suspension under control conditions (A), in the presence of Jasp (B) or LatB (C), and labeled for actin and E-cadherin. Merged images are shown in right panels. The images correspond to a medial transverse plane of the doublet. Dose-response curve of SF for 4-min Ecad doublets in medium containing Jasp (D) or LatB (E). (F) Mean SF for 30-s, 4- and 30-min doublets in the presence of 0.1 μM LatB (black bars), 0.1 μM Jasp (gray bars), or in control medium (white bars).
Mentions: We determined the effects of Jasp and LatB on actin by labeling Ecad doublets with an anti-actin mAb (not depicted) or phalloidin-TRITC (Fig. 6, B and C, respectively). Under control conditions, paired cells displayed a uniform distribution of surface E-cadherin and cortical actin over most of the cell with higher density colocalization of both molecules at the cell–cell interface (Fig. 6 A). Treatment with Jasp at 0.1 μM caused cortical actin and E-cadherin to redistribute in a nonuniform manner everywhere. However, at the contact zone both molecules were still noticeably colocalized. Jasp at 1 μM dramatically reduced the thickness of cortical actin, produced actin aggregates throughout the cytoplasm and eliminated the characteristic E-cadherin/actin colocalization at the cell–cell interface. Immunostaining of actin with mAb or phalloidin gave similar results and showed that, for doublets in suspension, Jasp at both 0.1 and 1 μM mainly induces a disorganization of the actin network reflecting the aggregation/polymerization activity of this drug described by Cramer (1999). LatB at 0.1 μM had no marked effect on the localization of E-cadherin and actin in paired cells in suspension (Fig. 6 C) but 1 μM LatB treatment induced the formation of large actin aggregates in the cytoplasm, E-cadherin clusters at the cell surface and higher levels of E-cadherin staining in the cytoplasm. FACS analysis demonstrated that LatB or Jasp, at concentrations up to 0.3 μM, does not affect E-cadherin expression at the cell surface (unpublished data).

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