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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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Redistribution of EcadGFP during cell–cell contact occurs in two stages and correlates with reorganization of the actin  cytoskeleton. Three 0.3-μm z-sections were collected from EcadGFP-expressing cells every 4.2 min at 0.5 micron/pixel at 6 sites.  (A) Combined stacks from two sites are shown (Contact 1 and  Contact 2). The age of each contact is displayed in min; the zero  time point defines when a stable cell–cell contact had formed.  Bar, 12 μm. The arrow points to a circumferential pattern of EcadGFP observed in single cells. (B and C) Immunofluorescence of  the same cells stained, after formaldehyde fixation, with  rhodamine phalloidin (actin; B) and mAb 3G8/CY5 (E-cadherin;  C). (D) EcadGFP organization shown in C is plotted as a function of fluorescence intensity (y axis, arbitrary fluorescence units)  vs. position in the contact (x axis, μm). The dashed gray lines running between C and D approximately register the edges of the  contact in the image and graph. (E and F) Triton X-100 extracted  wild-type MDCK cells (i.e., without EcadGFP) that had formed a  contact for <1 h (Contact 1) or >2 h (Contact 2) stained with  FITC-phalloidin and mAb 3G8/CY5, respectively. Bar, 10 μm.
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Figure 3: Redistribution of EcadGFP during cell–cell contact occurs in two stages and correlates with reorganization of the actin cytoskeleton. Three 0.3-μm z-sections were collected from EcadGFP-expressing cells every 4.2 min at 0.5 micron/pixel at 6 sites. (A) Combined stacks from two sites are shown (Contact 1 and Contact 2). The age of each contact is displayed in min; the zero time point defines when a stable cell–cell contact had formed. Bar, 12 μm. The arrow points to a circumferential pattern of EcadGFP observed in single cells. (B and C) Immunofluorescence of the same cells stained, after formaldehyde fixation, with rhodamine phalloidin (actin; B) and mAb 3G8/CY5 (E-cadherin; C). (D) EcadGFP organization shown in C is plotted as a function of fluorescence intensity (y axis, arbitrary fluorescence units) vs. position in the contact (x axis, μm). The dashed gray lines running between C and D approximately register the edges of the contact in the image and graph. (E and F) Triton X-100 extracted wild-type MDCK cells (i.e., without EcadGFP) that had formed a contact for <1 h (Contact 1) or >2 h (Contact 2) stained with FITC-phalloidin and mAb 3G8/CY5, respectively. Bar, 10 μm.

Mentions: To better understand the evolution of these distinct patterns of E-cadherin, the distribution of EcadGFP during development of cell–cell contacts was examined in multisite time-lapse confocal images taken over the course of 3 h (we initially focused on the formation of puncta and plaques during the first two stages of adhesion see below). The cells were then fixed and stained with phalloidin, (which labeled F-actin) and mAb 3G8 (which recognized the extracellular domain of endogenous E-cadherin and EcadGFP), and were imaged. Fig. 3 shows representative contacts from one time-lapse recording. Column 1 of Fig. 3 A shows the formation of a contact between two cells over 71 min. During cell–cell adhesion, EcadGFP fluorescence appeared at cell–cell contacts, and then increased in intensity with time and as the contact lengthened. The distribution of EcadGFP and endogenous E-cadherin were recorded retrospectively with mAb 3G8 (Fig. 3, 1C), showing that both proteins had coincident distributions as expected (see Fig. 1). Retrospective actin staining (Fig. 3, 1B) shows that circumferential actin cables were organized parallel to the cell–cell contact interface at this time.


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)

Redistribution of EcadGFP during cell–cell contact occurs in two stages and correlates with reorganization of the actin  cytoskeleton. Three 0.3-μm z-sections were collected from EcadGFP-expressing cells every 4.2 min at 0.5 micron/pixel at 6 sites.  (A) Combined stacks from two sites are shown (Contact 1 and  Contact 2). The age of each contact is displayed in min; the zero  time point defines when a stable cell–cell contact had formed.  Bar, 12 μm. The arrow points to a circumferential pattern of EcadGFP observed in single cells. (B and C) Immunofluorescence of  the same cells stained, after formaldehyde fixation, with  rhodamine phalloidin (actin; B) and mAb 3G8/CY5 (E-cadherin;  C). (D) EcadGFP organization shown in C is plotted as a function of fluorescence intensity (y axis, arbitrary fluorescence units)  vs. position in the contact (x axis, μm). The dashed gray lines running between C and D approximately register the edges of the  contact in the image and graph. (E and F) Triton X-100 extracted  wild-type MDCK cells (i.e., without EcadGFP) that had formed a  contact for <1 h (Contact 1) or >2 h (Contact 2) stained with  FITC-phalloidin and mAb 3G8/CY5, respectively. Bar, 10 μm.
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Figure 3: Redistribution of EcadGFP during cell–cell contact occurs in two stages and correlates with reorganization of the actin cytoskeleton. Three 0.3-μm z-sections were collected from EcadGFP-expressing cells every 4.2 min at 0.5 micron/pixel at 6 sites. (A) Combined stacks from two sites are shown (Contact 1 and Contact 2). The age of each contact is displayed in min; the zero time point defines when a stable cell–cell contact had formed. Bar, 12 μm. The arrow points to a circumferential pattern of EcadGFP observed in single cells. (B and C) Immunofluorescence of the same cells stained, after formaldehyde fixation, with rhodamine phalloidin (actin; B) and mAb 3G8/CY5 (E-cadherin; C). (D) EcadGFP organization shown in C is plotted as a function of fluorescence intensity (y axis, arbitrary fluorescence units) vs. position in the contact (x axis, μm). The dashed gray lines running between C and D approximately register the edges of the contact in the image and graph. (E and F) Triton X-100 extracted wild-type MDCK cells (i.e., without EcadGFP) that had formed a contact for <1 h (Contact 1) or >2 h (Contact 2) stained with FITC-phalloidin and mAb 3G8/CY5, respectively. Bar, 10 μm.
Mentions: To better understand the evolution of these distinct patterns of E-cadherin, the distribution of EcadGFP during development of cell–cell contacts was examined in multisite time-lapse confocal images taken over the course of 3 h (we initially focused on the formation of puncta and plaques during the first two stages of adhesion see below). The cells were then fixed and stained with phalloidin, (which labeled F-actin) and mAb 3G8 (which recognized the extracellular domain of endogenous E-cadherin and EcadGFP), and were imaged. Fig. 3 shows representative contacts from one time-lapse recording. Column 1 of Fig. 3 A shows the formation of a contact between two cells over 71 min. During cell–cell adhesion, EcadGFP fluorescence appeared at cell–cell contacts, and then increased in intensity with time and as the contact lengthened. The distribution of EcadGFP and endogenous E-cadherin were recorded retrospectively with mAb 3G8 (Fig. 3, 1C), showing that both proteins had coincident distributions as expected (see Fig. 1). Retrospective actin staining (Fig. 3, 1B) shows that circumferential actin cables were organized parallel to the cell–cell contact interface at this time.

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