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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 effect of dominant negative GTPase protein expression on SF. Distribution of GFP-tagged proteins in transfected Ecad cells producing GFP (B–D), and the Cdc42DN (F–H), RacDN (J–L), and RhoDN (N–P) before contact (B, F, J, and N), in 4-min doublets (C, G, K, and O) and in 30-min doublets (D, H, L, and P). Each row represents a series of real-time images of a doublet monitored by light transmission or epifluorescence microscopy before and at 4 and 30 min of contact. Q, SF measured for 4- and 30-min Ecad doublets producing either GFP (white bars), Cdc42DN (black bars), RacDN (dark gray bars), or RhoDN (light gray bars). (R) FACS analysis of transiently transfected Ecad cells, positive for GFP, Cdc42DN, RacDN, or RhoDN, and immunostained with an antibody directed against the extracellular domain of E-cadherin (FL2 channel).
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fig8: The effect of dominant negative GTPase protein expression on SF. Distribution of GFP-tagged proteins in transfected Ecad cells producing GFP (B–D), and the Cdc42DN (F–H), RacDN (J–L), and RhoDN (N–P) before contact (B, F, J, and N), in 4-min doublets (C, G, K, and O) and in 30-min doublets (D, H, L, and P). Each row represents a series of real-time images of a doublet monitored by light transmission or epifluorescence microscopy before and at 4 and 30 min of contact. Q, SF measured for 4- and 30-min Ecad doublets producing either GFP (white bars), Cdc42DN (black bars), RacDN (dark gray bars), or RhoDN (light gray bars). (R) FACS analysis of transiently transfected Ecad cells, positive for GFP, Cdc42DN, RacDN, or RhoDN, and immunostained with an antibody directed against the extracellular domain of E-cadherin (FL2 channel).

Mentions: We transiently transfected Ecad cells with pEGFPC1 alone (transfection control), or with vectors encoding the GFP-tagged constitutively inactive constructs, Cdc42DN, RacDN, and RhoDN or the GFP-tagged constitutively active constructs, Cdc42DA, RacDA, and RhoDA. In suspended isolated cells, GFP (Fig. 8 B) was distributed uniformly throughout the cytoplasm. For Cdc42DN (Fig. 8 F), RacDN (Fig. 8 J), RhoDN (Fig. 8 N), and RacDA (Fig. 9, G and H), we observed homogeneous fluorescence in the cytoplasm and more intense fluorescence close to the cell membrane. This distribution was also observed for Cdc42DA and RhoDA, but to a lesser extent (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)

The effect of dominant negative GTPase protein expression on SF. Distribution of GFP-tagged proteins in transfected Ecad cells producing GFP (B–D), and the Cdc42DN (F–H), RacDN (J–L), and RhoDN (N–P) before contact (B, F, J, and N), in 4-min doublets (C, G, K, and O) and in 30-min doublets (D, H, L, and P). Each row represents a series of real-time images of a doublet monitored by light transmission or epifluorescence microscopy before and at 4 and 30 min of contact. Q, SF measured for 4- and 30-min Ecad doublets producing either GFP (white bars), Cdc42DN (black bars), RacDN (dark gray bars), or RhoDN (light gray bars). (R) FACS analysis of transiently transfected Ecad cells, positive for GFP, Cdc42DN, RacDN, or RhoDN, and immunostained with an antibody directed against the extracellular domain of E-cadherin (FL2 channel).
© Copyright Policy
Related In: Results  -  Collection

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

fig8: The effect of dominant negative GTPase protein expression on SF. Distribution of GFP-tagged proteins in transfected Ecad cells producing GFP (B–D), and the Cdc42DN (F–H), RacDN (J–L), and RhoDN (N–P) before contact (B, F, J, and N), in 4-min doublets (C, G, K, and O) and in 30-min doublets (D, H, L, and P). Each row represents a series of real-time images of a doublet monitored by light transmission or epifluorescence microscopy before and at 4 and 30 min of contact. Q, SF measured for 4- and 30-min Ecad doublets producing either GFP (white bars), Cdc42DN (black bars), RacDN (dark gray bars), or RhoDN (light gray bars). (R) FACS analysis of transiently transfected Ecad cells, positive for GFP, Cdc42DN, RacDN, or RhoDN, and immunostained with an antibody directed against the extracellular domain of E-cadherin (FL2 channel).
Mentions: We transiently transfected Ecad cells with pEGFPC1 alone (transfection control), or with vectors encoding the GFP-tagged constitutively inactive constructs, Cdc42DN, RacDN, and RhoDN or the GFP-tagged constitutively active constructs, Cdc42DA, RacDA, and RhoDA. In suspended isolated cells, GFP (Fig. 8 B) was distributed uniformly throughout the cytoplasm. For Cdc42DN (Fig. 8 F), RacDN (Fig. 8 J), RhoDN (Fig. 8 N), and RacDA (Fig. 9, G and H), we observed homogeneous fluorescence in the cytoplasm and more intense fluorescence close to the cell membrane. This distribution was also observed for Cdc42DA and RhoDA, but to a lesser extent (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