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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 are formed and  stabilized along newly formed cell–cell contacts. (A) 16 time-lapse images of EcadGFP  cells taken from a sequence recorded every 1  min for 100 min at 0.11 μm/pixel; time in min  after formation of the contact is shown. Arrows follow a single punctum. (B) TIP scan of  all of the time-lapse images from the experiment represented in A. The contact was divided into 129 0.22-μm sections, and the maximum fluorescence intensity at 100 time  points was collected for a total of 12,900 data  points. The contact originates at 0 min and 0  μm. Small fluctuations in the apparent intensity of stable puncta are near the limits of instrumental noise sources such as laser output  fluctuations and noise processes in the photomultiplier tube detector. (C) Double immunofluorescence of the same contact extracted  with Triton X-100, fixed with formaldehyde,  and stained with rhodamine phalloidin and  E-cadherin mAb 3G8/CY5. The arrows in all  panels point to the same punctum. Bars: (A)  10 μm; (C) 5 μm. (B) 0–210 gray scale fluorescence intensity units divided into 15 colors.
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Figure 4: EcadGFP puncta are formed and stabilized along newly formed cell–cell contacts. (A) 16 time-lapse images of EcadGFP cells taken from a sequence recorded every 1 min for 100 min at 0.11 μm/pixel; time in min after formation of the contact is shown. Arrows follow a single punctum. (B) TIP scan of all of the time-lapse images from the experiment represented in A. The contact was divided into 129 0.22-μm sections, and the maximum fluorescence intensity at 100 time points was collected for a total of 12,900 data points. The contact originates at 0 min and 0 μm. Small fluctuations in the apparent intensity of stable puncta are near the limits of instrumental noise sources such as laser output fluctuations and noise processes in the photomultiplier tube detector. (C) Double immunofluorescence of the same contact extracted with Triton X-100, fixed with formaldehyde, and stained with rhodamine phalloidin and E-cadherin mAb 3G8/CY5. The arrows in all panels point to the same punctum. Bars: (A) 10 μm; (C) 5 μm. (B) 0–210 gray scale fluorescence intensity units divided into 15 colors.

Mentions: To gain information about the genesis, lifetime, and position of EcadGFP during initiation of contact formation, a single field of EcadGFP-expressing cells was imaged rapidly at high resolution. Fig. 4 A shows representative images from one of these time-lapse recordings. An arrow follows the position of a bright EcadGFP fluorescent punctum at the cell–cell interface. To provide an objective nonbiased format for the quantitative representation of dynamic data like that illustrated in Fig. 4 A, we developed the type of representation shown in Fig. 4 B. The fluorescence intensity profiles along the length of the contact (e.g., Fig. 3 D) were color-coded and combined for each time-lapse frame to provide a color map of EcadGFP intensity distribution along the length of the contact as the contact lengthened. We term such graphs TIP scans. By providing a clear representation of time-dependent changes in EcadGFP fluorescence along the cell–cell contact interface, TIP scans make it relatively easy to discern the organization of EcadGFP during contact formation. Background fluorescence in the TIP scan is contributed by overlapping regions of plasma membrane. Areas of the contact that are brighter than the background cell fluorescence correspond to brighter clusters of EcadGFP. The TIP scan in Fig. 4 B shows that the contact in Fig. 4 A grew to a length of ∼12 μm in ∼20 min. The contact then grew more slowly to reach a length of ∼35 μm after 90 min.


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 are formed and  stabilized along newly formed cell–cell contacts. (A) 16 time-lapse images of EcadGFP  cells taken from a sequence recorded every 1  min for 100 min at 0.11 μm/pixel; time in min  after formation of the contact is shown. Arrows follow a single punctum. (B) TIP scan of  all of the time-lapse images from the experiment represented in A. The contact was divided into 129 0.22-μm sections, and the maximum fluorescence intensity at 100 time  points was collected for a total of 12,900 data  points. The contact originates at 0 min and 0  μm. Small fluctuations in the apparent intensity of stable puncta are near the limits of instrumental noise sources such as laser output  fluctuations and noise processes in the photomultiplier tube detector. (C) Double immunofluorescence of the same contact extracted  with Triton X-100, fixed with formaldehyde,  and stained with rhodamine phalloidin and  E-cadherin mAb 3G8/CY5. The arrows in all  panels point to the same punctum. Bars: (A)  10 μm; (C) 5 μm. (B) 0–210 gray scale fluorescence intensity units divided into 15 colors.
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Figure 4: EcadGFP puncta are formed and stabilized along newly formed cell–cell contacts. (A) 16 time-lapse images of EcadGFP cells taken from a sequence recorded every 1 min for 100 min at 0.11 μm/pixel; time in min after formation of the contact is shown. Arrows follow a single punctum. (B) TIP scan of all of the time-lapse images from the experiment represented in A. The contact was divided into 129 0.22-μm sections, and the maximum fluorescence intensity at 100 time points was collected for a total of 12,900 data points. The contact originates at 0 min and 0 μm. Small fluctuations in the apparent intensity of stable puncta are near the limits of instrumental noise sources such as laser output fluctuations and noise processes in the photomultiplier tube detector. (C) Double immunofluorescence of the same contact extracted with Triton X-100, fixed with formaldehyde, and stained with rhodamine phalloidin and E-cadherin mAb 3G8/CY5. The arrows in all panels point to the same punctum. Bars: (A) 10 μm; (C) 5 μm. (B) 0–210 gray scale fluorescence intensity units divided into 15 colors.
Mentions: To gain information about the genesis, lifetime, and position of EcadGFP during initiation of contact formation, a single field of EcadGFP-expressing cells was imaged rapidly at high resolution. Fig. 4 A shows representative images from one of these time-lapse recordings. An arrow follows the position of a bright EcadGFP fluorescent punctum at the cell–cell interface. To provide an objective nonbiased format for the quantitative representation of dynamic data like that illustrated in Fig. 4 A, we developed the type of representation shown in Fig. 4 B. The fluorescence intensity profiles along the length of the contact (e.g., Fig. 3 D) were color-coded and combined for each time-lapse frame to provide a color map of EcadGFP intensity distribution along the length of the contact as the contact lengthened. We term such graphs TIP scans. By providing a clear representation of time-dependent changes in EcadGFP fluorescence along the cell–cell contact interface, TIP scans make it relatively easy to discern the organization of EcadGFP during contact formation. Background fluorescence in the TIP scan is contributed by overlapping regions of plasma membrane. Areas of the contact that are brighter than the background cell fluorescence correspond to brighter clusters of EcadGFP. The TIP scan in Fig. 4 B shows that the contact in Fig. 4 A grew to a length of ∼12 μm in ∼20 min. The contact then grew more slowly to reach a length of ∼35 μm after 90 min.

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