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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 puncta cluster into plaques during transition  between early and late stages of adhesion. (A) Representative  images of a time-lapse sequence taken at 0.8 Hz for 300 s at 0.12  μm/pixel in a region of the cell–cell contact in which a plaque is  developing. Time is in min; arrows point to individual puncta;  bar, 2 μm. (B) Quantitative fluorescence intensities of EcadGFP.  The average (gray circles) and maximum (black diamonds) intensities in a 20-μm2 region surrounding a developing plaque area  are plotted over a 40-min period. The average fluorescence intensity of the same number of EcadGFP in a fixed area is constant  regardless of its distribution. However, the maximum fluorescence intensity increases as EcadGFP clusters into a smaller  region within that fixed area. For details, see Materials and  Methods.
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Figure 6: EcadGFP puncta cluster into plaques during transition between early and late stages of adhesion. (A) Representative images of a time-lapse sequence taken at 0.8 Hz for 300 s at 0.12 μm/pixel in a region of the cell–cell contact in which a plaque is developing. Time is in min; arrows point to individual puncta; bar, 2 μm. (B) Quantitative fluorescence intensities of EcadGFP. The average (gray circles) and maximum (black diamonds) intensities in a 20-μm2 region surrounding a developing plaque area are plotted over a 40-min period. The average fluorescence intensity of the same number of EcadGFP in a fixed area is constant regardless of its distribution. However, the maximum fluorescence intensity increases as EcadGFP clusters into a smaller region within that fixed area. For details, see Materials and Methods.

Mentions: The gradual increase in the amount of EcadGFP in these plaques might be the result of de novo clustering of EcadGFP around new actin filaments exposed at the margins of the cell–cell contact, or from the aggregation and migration of puncta that had preformed along the length of the contact. To distinguish between these two possibilities, time-lapse images of EcadGFP plaques were recorded rapidly for 300 s at high resolution. Fig. 6 A shows a representative montage of images in which a plaque was observed forming from an area of membrane that contained many small puncta (Fig. 6 A, arrowheads). The small puncta clearly merged together over time to form the larger plaque. The fluorescence intensity of the plaque increased concomitantly with the disappearance of individual puncta. We quantified changes in fluorescence intensity and plotted maximum fluorescence intensity values (i.e., the density of EcadGFP) and average fluorescence intensity values (i.e., the total amount of EcadGFP) in large (>10 μm diameter ) regions surrounding the edge of the developing contact (Fig. 6 B). It is clear that the peak density (Fig. 6 B, black diamonds), but not the total amount (Fig. 6 B, gray circles) of EcadGFP increased in the region of the membrane containing the forming plaque. Thus, plaques most likely to arise by lateral clustering of a subset of EcadGFP puncta already formed along the cell–cell contact, and are perhaps supplemented by recruitment of additional EcadGFP molecules in the area of plaque formation.


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 puncta cluster into plaques during transition  between early and late stages of adhesion. (A) Representative  images of a time-lapse sequence taken at 0.8 Hz for 300 s at 0.12  μm/pixel in a region of the cell–cell contact in which a plaque is  developing. Time is in min; arrows point to individual puncta;  bar, 2 μm. (B) Quantitative fluorescence intensities of EcadGFP.  The average (gray circles) and maximum (black diamonds) intensities in a 20-μm2 region surrounding a developing plaque area  are plotted over a 40-min period. The average fluorescence intensity of the same number of EcadGFP in a fixed area is constant  regardless of its distribution. However, the maximum fluorescence intensity increases as EcadGFP clusters into a smaller  region within that fixed area. For details, see Materials and  Methods.
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Related In: Results  -  Collection

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Figure 6: EcadGFP puncta cluster into plaques during transition between early and late stages of adhesion. (A) Representative images of a time-lapse sequence taken at 0.8 Hz for 300 s at 0.12 μm/pixel in a region of the cell–cell contact in which a plaque is developing. Time is in min; arrows point to individual puncta; bar, 2 μm. (B) Quantitative fluorescence intensities of EcadGFP. The average (gray circles) and maximum (black diamonds) intensities in a 20-μm2 region surrounding a developing plaque area are plotted over a 40-min period. The average fluorescence intensity of the same number of EcadGFP in a fixed area is constant regardless of its distribution. However, the maximum fluorescence intensity increases as EcadGFP clusters into a smaller region within that fixed area. For details, see Materials and Methods.
Mentions: The gradual increase in the amount of EcadGFP in these plaques might be the result of de novo clustering of EcadGFP around new actin filaments exposed at the margins of the cell–cell contact, or from the aggregation and migration of puncta that had preformed along the length of the contact. To distinguish between these two possibilities, time-lapse images of EcadGFP plaques were recorded rapidly for 300 s at high resolution. Fig. 6 A shows a representative montage of images in which a plaque was observed forming from an area of membrane that contained many small puncta (Fig. 6 A, arrowheads). The small puncta clearly merged together over time to form the larger plaque. The fluorescence intensity of the plaque increased concomitantly with the disappearance of individual puncta. We quantified changes in fluorescence intensity and plotted maximum fluorescence intensity values (i.e., the density of EcadGFP) and average fluorescence intensity values (i.e., the total amount of EcadGFP) in large (>10 μm diameter ) regions surrounding the edge of the developing contact (Fig. 6 B). It is clear that the peak density (Fig. 6 B, black diamonds), but not the total amount (Fig. 6 B, gray circles) of EcadGFP increased in the region of the membrane containing the forming plaque. Thus, plaques most likely to arise by lateral clustering of a subset of EcadGFP puncta already formed along the cell–cell contact, and are perhaps supplemented by recruitment of additional EcadGFP molecules in the area of plaque formation.

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