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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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Two large plaques of EcadGFP  form and move to the edges of the cell–cell  contact. (A) 16 time-lapse images of EcadGFP cells taken from a sequence recorded every 2 min for 2.7 h at 0.23 μm/pixel. Arrows  follow a single plaque. (B) TIP scan of all of  the time-lapse images from the experiment  represented in A. The contact was divided  into 101, 0.46-μm sections, and the fluorescence intensity at 85 time points was collected  for a total of 8,585 data points. The contact  originates at 0 min and 0 μm. Note that the  TIP scan at this reduced resolution shows a  relatively homogeneous distribution of EcadGFP within the contact during the first hour,  whereas the TIP scan at a higher resolution  revealed individual punctum (see Fig. 4). (C)  Double immunofluorescence of the same  contact stained with rhodamine phalloidin  and E-cadherin mAb 3G8/CY5. The arrows  in all panels point to the same plaque. (B) 0– 151 gray scale fluorescence intensity units divided into 15 colors. Bars: (A) 10 μm and (C)  5 μm.
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Figure 5: Two large plaques of EcadGFP form and move to the edges of the cell–cell contact. (A) 16 time-lapse images of EcadGFP cells taken from a sequence recorded every 2 min for 2.7 h at 0.23 μm/pixel. Arrows follow a single plaque. (B) TIP scan of all of the time-lapse images from the experiment represented in A. The contact was divided into 101, 0.46-μm sections, and the fluorescence intensity at 85 time points was collected for a total of 8,585 data points. The contact originates at 0 min and 0 μm. Note that the TIP scan at this reduced resolution shows a relatively homogeneous distribution of EcadGFP within the contact during the first hour, whereas the TIP scan at a higher resolution revealed individual punctum (see Fig. 4). (C) Double immunofluorescence of the same contact stained with rhodamine phalloidin and E-cadherin mAb 3G8/CY5. The arrows in all panels point to the same plaque. (B) 0– 151 gray scale fluorescence intensity units divided into 15 colors. Bars: (A) 10 μm and (C) 5 μm.

Mentions: Next, we asked how early E-cadherin puncta are reorganized with actin to further strengthen cell–cell adhesion, and then to cause condensation of cells into multicell colonies. Over longer times (>2 h), EcadGFP and endogenous E-cadherin became organized into large plaques at the margins of the contact (Fig. 2 and Fig. 3, column 2). Fig. 5 A shows representative images from a longer time-lapse experiment. After ∼1.5 h, two regions of increasing EcadGFP fluorescence appeared and migrated out with the edges of the contact at velocities of up to 0.5 μm/min (Fig. 5). These regions gradually gained up to 10× the average punctum fluorescence intensity over the course of 1 h, in contrast to a punctum that reached maximum fluorescence intensity within 30 min of formation. Approximately 2.5 h after contact nucleation, EcadGFP was heavily concentrated in discrete fluorescent plaques at the margins of the contact (Fig. 5, A and B, arrows). The region of the contact between the two plaques retained a thin line of EcadGFP intensity (compare to Fig. 3, column 2B). Comparison of the last live EcadGFP images with the retrospective immunofluorescence of E-cadherin and actin (Fig. 5 C) shows that EcadGFP plaques were resistant to extraction with Triton X-100, and were sites at which circumferential actin cables terminated.


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)

Two large plaques of EcadGFP  form and move to the edges of the cell–cell  contact. (A) 16 time-lapse images of EcadGFP cells taken from a sequence recorded every 2 min for 2.7 h at 0.23 μm/pixel. Arrows  follow a single plaque. (B) TIP scan of all of  the time-lapse images from the experiment  represented in A. The contact was divided  into 101, 0.46-μm sections, and the fluorescence intensity at 85 time points was collected  for a total of 8,585 data points. The contact  originates at 0 min and 0 μm. Note that the  TIP scan at this reduced resolution shows a  relatively homogeneous distribution of EcadGFP within the contact during the first hour,  whereas the TIP scan at a higher resolution  revealed individual punctum (see Fig. 4). (C)  Double immunofluorescence of the same  contact stained with rhodamine phalloidin  and E-cadherin mAb 3G8/CY5. The arrows  in all panels point to the same plaque. (B) 0– 151 gray scale fluorescence intensity units divided into 15 colors. Bars: (A) 10 μm and (C)  5 μm.
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Related In: Results  -  Collection

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Figure 5: Two large plaques of EcadGFP form and move to the edges of the cell–cell contact. (A) 16 time-lapse images of EcadGFP cells taken from a sequence recorded every 2 min for 2.7 h at 0.23 μm/pixel. Arrows follow a single plaque. (B) TIP scan of all of the time-lapse images from the experiment represented in A. The contact was divided into 101, 0.46-μm sections, and the fluorescence intensity at 85 time points was collected for a total of 8,585 data points. The contact originates at 0 min and 0 μm. Note that the TIP scan at this reduced resolution shows a relatively homogeneous distribution of EcadGFP within the contact during the first hour, whereas the TIP scan at a higher resolution revealed individual punctum (see Fig. 4). (C) Double immunofluorescence of the same contact stained with rhodamine phalloidin and E-cadherin mAb 3G8/CY5. The arrows in all panels point to the same plaque. (B) 0– 151 gray scale fluorescence intensity units divided into 15 colors. Bars: (A) 10 μm and (C) 5 μm.
Mentions: Next, we asked how early E-cadherin puncta are reorganized with actin to further strengthen cell–cell adhesion, and then to cause condensation of cells into multicell colonies. Over longer times (>2 h), EcadGFP and endogenous E-cadherin became organized into large plaques at the margins of the contact (Fig. 2 and Fig. 3, column 2). Fig. 5 A shows representative images from a longer time-lapse experiment. After ∼1.5 h, two regions of increasing EcadGFP fluorescence appeared and migrated out with the edges of the contact at velocities of up to 0.5 μm/min (Fig. 5). These regions gradually gained up to 10× the average punctum fluorescence intensity over the course of 1 h, in contrast to a punctum that reached maximum fluorescence intensity within 30 min of formation. Approximately 2.5 h after contact nucleation, EcadGFP was heavily concentrated in discrete fluorescent plaques at the margins of the contact (Fig. 5, A and B, arrows). The region of the contact between the two plaques retained a thin line of EcadGFP intensity (compare to Fig. 3, column 2B). Comparison of the last live EcadGFP images with the retrospective immunofluorescence of E-cadherin and actin (Fig. 5 C) shows that EcadGFP plaques were resistant to extraction with Triton X-100, and were sites at which circumferential actin cables terminated.

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