Limits...
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.

Show MeSH

Related in: MedlinePlus

Cytochalasin D selectively disassembles new cell–cell  contacts. Representative images of a time-lapse sequence taken  at 1 frame/2 min for 2 h at 0.4 μm/pixel before and after adding  2 μM CD. (A) 14 min before CD; (B) 1 min before CD; (C) 30  min after CD; (D) 60 min after CD. Immunofluorescence of the  same area is shown using rhodamine phalloidin (E) or β-catenin/ CY5 (F). The arrows in D–F point to CD-induced EcadGFP clusters; bar, 10 μm. (G) The number of cells that were in contact before the time-lapse experiment began (>60 min old), and those  that made contact during the imaging experiment (<60 min old)  were counted and the totals shown for three independent experiments (black bars). The number of those contacts that disassembled within 1 h after CD treatment was determined (striped bars).  The percentage of cell–cell contacts disassembled by CD treatment is 14% for old contacts and 73% for new contacts.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132880&req=5

Figure 7: Cytochalasin D selectively disassembles new cell–cell contacts. Representative images of a time-lapse sequence taken at 1 frame/2 min for 2 h at 0.4 μm/pixel before and after adding 2 μM CD. (A) 14 min before CD; (B) 1 min before CD; (C) 30 min after CD; (D) 60 min after CD. Immunofluorescence of the same area is shown using rhodamine phalloidin (E) or β-catenin/ CY5 (F). The arrows in D–F point to CD-induced EcadGFP clusters; bar, 10 μm. (G) The number of cells that were in contact before the time-lapse experiment began (>60 min old), and those that made contact during the imaging experiment (<60 min old) were counted and the totals shown for three independent experiments (black bars). The number of those contacts that disassembled within 1 h after CD treatment was determined (striped bars). The percentage of cell–cell contacts disassembled by CD treatment is 14% for old contacts and 73% for new contacts.

Mentions: To obtain information about the role of actin during the formation and stabilization of cell–cell contacts, cells were treated with the actin-capping agent cytochalasin D (CD). After multisite time-lapse recording for 1 h, EcadGFP cells were treated with 2 μM CD. New cell–cell contacts did not form in the presence of CD (data not shown). Furthermore, young cell–cell contacts (Fig. 7, A and B) disassembled upon addition of CD (Fig. 7, C and D). Analogous to an intact monolayer of cells (Hirano et al., 1987), cell–cell contacts within small colonies that were >1 h old did not disassemble during CD treatment. Cells that were in contact for <1 h (Fig. 7 A, upper right and lower left) rounded after CD treatment (compare Fig. 7, A and D). Greater than 70% of 19 cell–cell contacts that were <1 h old disassembled after treatment with CD (Fig. 7 G). In contrast, >15% of 48 contacts that were >1 h old disassembled (Fig. 7 G). During CD treatment, EcadGFP formed aggregates that coincided with irregularities in the DIC images (Fig. 7 D, arrow). These aggregates colocalized with actin and β-catenin (Fig. 7, E and F) and areas that have been shown to be enriched in the barbed ends of actin filaments (Verkhousky et al., 1997), suggesting that the cadherin/catenin complex may associate with the barbed ends of actin filaments. Furthermore, these data indicate that capping the barbed ends of actin filaments with CD disrupts the ability of E-cadherin puncta, but not plaques to maintain the integrity of cell–cell contacts.


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)

Cytochalasin D selectively disassembles new cell–cell  contacts. Representative images of a time-lapse sequence taken  at 1 frame/2 min for 2 h at 0.4 μm/pixel before and after adding  2 μM CD. (A) 14 min before CD; (B) 1 min before CD; (C) 30  min after CD; (D) 60 min after CD. Immunofluorescence of the  same area is shown using rhodamine phalloidin (E) or β-catenin/ CY5 (F). The arrows in D–F point to CD-induced EcadGFP clusters; bar, 10 μm. (G) The number of cells that were in contact before the time-lapse experiment began (>60 min old), and those  that made contact during the imaging experiment (<60 min old)  were counted and the totals shown for three independent experiments (black bars). The number of those contacts that disassembled within 1 h after CD treatment was determined (striped bars).  The percentage of cell–cell contacts disassembled by CD treatment is 14% for old contacts and 73% for new contacts.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132880&req=5

Figure 7: Cytochalasin D selectively disassembles new cell–cell contacts. Representative images of a time-lapse sequence taken at 1 frame/2 min for 2 h at 0.4 μm/pixel before and after adding 2 μM CD. (A) 14 min before CD; (B) 1 min before CD; (C) 30 min after CD; (D) 60 min after CD. Immunofluorescence of the same area is shown using rhodamine phalloidin (E) or β-catenin/ CY5 (F). The arrows in D–F point to CD-induced EcadGFP clusters; bar, 10 μm. (G) The number of cells that were in contact before the time-lapse experiment began (>60 min old), and those that made contact during the imaging experiment (<60 min old) were counted and the totals shown for three independent experiments (black bars). The number of those contacts that disassembled within 1 h after CD treatment was determined (striped bars). The percentage of cell–cell contacts disassembled by CD treatment is 14% for old contacts and 73% for new contacts.
Mentions: To obtain information about the role of actin during the formation and stabilization of cell–cell contacts, cells were treated with the actin-capping agent cytochalasin D (CD). After multisite time-lapse recording for 1 h, EcadGFP cells were treated with 2 μM CD. New cell–cell contacts did not form in the presence of CD (data not shown). Furthermore, young cell–cell contacts (Fig. 7, A and B) disassembled upon addition of CD (Fig. 7, C and D). Analogous to an intact monolayer of cells (Hirano et al., 1987), cell–cell contacts within small colonies that were >1 h old did not disassemble during CD treatment. Cells that were in contact for <1 h (Fig. 7 A, upper right and lower left) rounded after CD treatment (compare Fig. 7, A and D). Greater than 70% of 19 cell–cell contacts that were <1 h old disassembled after treatment with CD (Fig. 7 G). In contrast, >15% of 48 contacts that were >1 h old disassembled (Fig. 7 G). During CD treatment, EcadGFP formed aggregates that coincided with irregularities in the DIC images (Fig. 7 D, arrow). These aggregates colocalized with actin and β-catenin (Fig. 7, E and F) and areas that have been shown to be enriched in the barbed ends of actin filaments (Verkhousky et al., 1997), suggesting that the cadherin/catenin complex may associate with the barbed ends of actin filaments. Furthermore, these data indicate that capping the barbed ends of actin filaments with CD disrupts the ability of E-cadherin puncta, but not plaques to maintain the integrity of cell–cell contacts.

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