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Mechanisms of epithelial cell-cell adhesion and cell compaction revealed by high-resolution tracking of E-cadherin-green fluorescent protein.

Adams CL, Chen YT, Smith SJ, Nelson WJ - J. Cell Biol. (1998)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5345, USA.

ABSTRACT
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.

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EcadGFP has properties similar to those of endogenous E-cadherin. (A and B) Catenins were coimmunoprecipitated with EcadGFP from HEK 293 cells transiently transfected  with EcadGFP (A) or stably transfected MDCK cells (B). Cells  were labeled with [35S]Met/Cys for 24 h before immunoprecipitation. (A) Proteins in cadherin immunoprecipitates are compared  among mock-transfected HEK 293 cells (No DNA), HEK 293  cells transfected with canine E-cadherin (Ecad), and HEK 293  cells transfected with EcadGFP, and (B) between untransfected  MDCK cells (No DNA) and MDCK cells stably transfected with  EcadGFP. (C) EcadGFP fluorescence and β-catenin immunofluorescence from HEK 293 cells transiently transfected with EcadGFP. Bar, 30 μm. (D) Preferential delivery of newly synthesized  EcadGFP to the basal-lateral plasma membrane of fully polarized MDCK cells stably transfected with EcadGFP, or of endogenous E-cadherin in untransfected cells (No DNA); A, apical  membrane; B, basal-lateral membrane. (E) Phase contrast images of L-cells and L-cells expressing EcadGFP after 18 h of aggregation in suspension culture in the presence or absence of extracellular Ca2+. Bar, 60 μm.
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Figure 1: EcadGFP has properties similar to those of endogenous E-cadherin. (A and B) Catenins were coimmunoprecipitated with EcadGFP from HEK 293 cells transiently transfected with EcadGFP (A) or stably transfected MDCK cells (B). Cells were labeled with [35S]Met/Cys for 24 h before immunoprecipitation. (A) Proteins in cadherin immunoprecipitates are compared among mock-transfected HEK 293 cells (No DNA), HEK 293 cells transfected with canine E-cadherin (Ecad), and HEK 293 cells transfected with EcadGFP, and (B) between untransfected MDCK cells (No DNA) and MDCK cells stably transfected with EcadGFP. (C) EcadGFP fluorescence and β-catenin immunofluorescence from HEK 293 cells transiently transfected with EcadGFP. Bar, 30 μm. (D) Preferential delivery of newly synthesized EcadGFP to the basal-lateral plasma membrane of fully polarized MDCK cells stably transfected with EcadGFP, or of endogenous E-cadherin in untransfected cells (No DNA); A, apical membrane; B, basal-lateral membrane. (E) Phase contrast images of L-cells and L-cells expressing EcadGFP after 18 h of aggregation in suspension culture in the presence or absence of extracellular Ca2+. Bar, 60 μm.

Mentions: To examine directly the dynamics of E-cadherin in living cells, we constructed a fusion protein composed of full-length canine E-cadherin fused at the carboxyl terminus to GFP (EcadGFP). EcadGFP was expressed in MDCK cells (Fig. 1, B and D), HEK 293 EBNA cells (Fig. 1, A and C), and L cells (Fig. 1 E). In all cell types, EcadGFP had an apparent molecular mass of ∼150 kD (Fig. 1, A and B) consistent with the combined molecular masses of the fused proteins; a minor protein band of ∼160 kD was the likely precursor. Expression of endogenous E-cadherin in MDCK cells was suppressed to some extent in the presence of EcadGFP (Fig. 1 B); thus, EcadGFP contributes significantly to cell–cell adhesion in these MDCK cells. Analysis of EcadGFP-immunoprecipitated protein complexes showed the presence of three additional bands at ∼102, 98, and 86 kD (Fig. 1 B), corresponding to the molecular weights of α-, β-, and γ-catenin (plakoglobin), respectively. The stoichiometry of the EcadGFP/catenin complex was similar to that of the endogenous E-cadherin/ catenin complex (see Fig. 1, A and B).


Mechanisms of epithelial cell-cell adhesion and cell compaction revealed by high-resolution tracking of E-cadherin-green fluorescent protein.

Adams CL, Chen YT, Smith SJ, Nelson WJ - J. Cell Biol. (1998)

EcadGFP has properties similar to those of endogenous E-cadherin. (A and B) Catenins were coimmunoprecipitated with EcadGFP from HEK 293 cells transiently transfected  with EcadGFP (A) or stably transfected MDCK cells (B). Cells  were labeled with [35S]Met/Cys for 24 h before immunoprecipitation. (A) Proteins in cadherin immunoprecipitates are compared  among mock-transfected HEK 293 cells (No DNA), HEK 293  cells transfected with canine E-cadherin (Ecad), and HEK 293  cells transfected with EcadGFP, and (B) between untransfected  MDCK cells (No DNA) and MDCK cells stably transfected with  EcadGFP. (C) EcadGFP fluorescence and β-catenin immunofluorescence from HEK 293 cells transiently transfected with EcadGFP. Bar, 30 μm. (D) Preferential delivery of newly synthesized  EcadGFP to the basal-lateral plasma membrane of fully polarized MDCK cells stably transfected with EcadGFP, or of endogenous E-cadherin in untransfected cells (No DNA); A, apical  membrane; B, basal-lateral membrane. (E) Phase contrast images of L-cells and L-cells expressing EcadGFP after 18 h of aggregation in suspension culture in the presence or absence of extracellular Ca2+. Bar, 60 μm.
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Related In: Results  -  Collection

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Figure 1: EcadGFP has properties similar to those of endogenous E-cadherin. (A and B) Catenins were coimmunoprecipitated with EcadGFP from HEK 293 cells transiently transfected with EcadGFP (A) or stably transfected MDCK cells (B). Cells were labeled with [35S]Met/Cys for 24 h before immunoprecipitation. (A) Proteins in cadherin immunoprecipitates are compared among mock-transfected HEK 293 cells (No DNA), HEK 293 cells transfected with canine E-cadherin (Ecad), and HEK 293 cells transfected with EcadGFP, and (B) between untransfected MDCK cells (No DNA) and MDCK cells stably transfected with EcadGFP. (C) EcadGFP fluorescence and β-catenin immunofluorescence from HEK 293 cells transiently transfected with EcadGFP. Bar, 30 μm. (D) Preferential delivery of newly synthesized EcadGFP to the basal-lateral plasma membrane of fully polarized MDCK cells stably transfected with EcadGFP, or of endogenous E-cadherin in untransfected cells (No DNA); A, apical membrane; B, basal-lateral membrane. (E) Phase contrast images of L-cells and L-cells expressing EcadGFP after 18 h of aggregation in suspension culture in the presence or absence of extracellular Ca2+. Bar, 60 μm.
Mentions: To examine directly the dynamics of E-cadherin in living cells, we constructed a fusion protein composed of full-length canine E-cadherin fused at the carboxyl terminus to GFP (EcadGFP). EcadGFP was expressed in MDCK cells (Fig. 1, B and D), HEK 293 EBNA cells (Fig. 1, A and C), and L cells (Fig. 1 E). In all cell types, EcadGFP had an apparent molecular mass of ∼150 kD (Fig. 1, A and B) consistent with the combined molecular masses of the fused proteins; a minor protein band of ∼160 kD was the likely precursor. Expression of endogenous E-cadherin in MDCK cells was suppressed to some extent in the presence of EcadGFP (Fig. 1 B); thus, EcadGFP contributes significantly to cell–cell adhesion in these MDCK cells. Analysis of EcadGFP-immunoprecipitated protein complexes showed the presence of three additional bands at ∼102, 98, and 86 kD (Fig. 1 B), corresponding to the molecular weights of α-, β-, and γ-catenin (plakoglobin), respectively. The stoichiometry of the EcadGFP/catenin complex was similar to that of the endogenous E-cadherin/ catenin complex (see Fig. 1, A and B).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5345, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus