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

Photobleach-recovery analysis shows a highly  mobile pool of EcadGFP coalesces into immobile puncta.  A shows a live cell before  and after photobleaching.  The box indicates where the  cell was photobleached. The  arrow points to an area that  formed a contact during the  photobleach. The cells were  fixed in formaldehyde and  stained with phalloidin and  mAb 3G8. B shows the fluorescence recovery curves of a  single noncontacting cell in  which half of the cell was  photobleached (blue). EcadGFP fluorescence of the nonbleached region (red) and  the entire cell (green) was  monitored during recovery.  Notice that the EcadGFP fluorescence values equalize in  the photobleached and nonphotobleached areas. C shows  the first 3 min of photobleach-recovery data of noncontacting membrane regions photobleached with a  5.8-μm (pink) and 3-μm  (black) circle. The relative  fluorescence is scaled between the fluorescence intensity just after bleaching and  equilibrium. The lines show  the theoretical recovery  curves for each region with a  diffusion coefficient of 3 ×  10−10 cm2/s. Note that the  smaller photobleach circle (black line) recovers more quickly. D shows images taken before, 0.1 min after, and 10 min after photobleaching a 5.8-μm-diameter circle in a region of membrane not involved in cell–cell contact (Membrane), a region of membrane in a  <15-min-old contact (New contact), a region of membrane in the middle of a <60-min-old contact (Puncta), and a membrane at the  edge of a >2-h-old contact (Plaque). For each experiment, 300 images were collected every 3.2 s at 0.11 μm/pixel. The circles mark the  photobleach region, and the colors correspond to the recovery curves shown in C (pink) and E. E shows photobleach-recovery data for  the bleached contact regions identified in D. The relative fluorescence is scaled to the pre-bleach intensity value. The mobile fraction  of EcadGFP in the new contact (blue), puncta (green), and plaque (orange) is 100, 50, and <10%, respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132880&req=5

Figure 8: Photobleach-recovery analysis shows a highly mobile pool of EcadGFP coalesces into immobile puncta. A shows a live cell before and after photobleaching. The box indicates where the cell was photobleached. The arrow points to an area that formed a contact during the photobleach. The cells were fixed in formaldehyde and stained with phalloidin and mAb 3G8. B shows the fluorescence recovery curves of a single noncontacting cell in which half of the cell was photobleached (blue). EcadGFP fluorescence of the nonbleached region (red) and the entire cell (green) was monitored during recovery. Notice that the EcadGFP fluorescence values equalize in the photobleached and nonphotobleached areas. C shows the first 3 min of photobleach-recovery data of noncontacting membrane regions photobleached with a 5.8-μm (pink) and 3-μm (black) circle. The relative fluorescence is scaled between the fluorescence intensity just after bleaching and equilibrium. The lines show the theoretical recovery curves for each region with a diffusion coefficient of 3 × 10−10 cm2/s. Note that the smaller photobleach circle (black line) recovers more quickly. D shows images taken before, 0.1 min after, and 10 min after photobleaching a 5.8-μm-diameter circle in a region of membrane not involved in cell–cell contact (Membrane), a region of membrane in a <15-min-old contact (New contact), a region of membrane in the middle of a <60-min-old contact (Puncta), and a membrane at the edge of a >2-h-old contact (Plaque). For each experiment, 300 images were collected every 3.2 s at 0.11 μm/pixel. The circles mark the photobleach region, and the colors correspond to the recovery curves shown in C (pink) and E. E shows photobleach-recovery data for the bleached contact regions identified in D. The relative fluorescence is scaled to the pre-bleach intensity value. The mobile fraction of EcadGFP in the new contact (blue), puncta (green), and plaque (orange) is 100, 50, and <10%, respectively.

Mentions: To gain further insight into the assembly dynamics of E-cadherin puncta and plaques, we developed a photobleaching-recovery method to measure the diffusion coefficient, mobile fraction, and redistribution of EcadGFP during different stages of contact development (see Materials and Methods). Photobleaching of cell–cell contacts neither disrupts the organization of adhering plasma membranes, induces retraction of membranes, nor changes membrane movements or dynamics (Fig. 8 A). Fig. 8 A shows the DIC and EcadGFP images for a live cell before and after photobleaching. After photobleaching, the cells were immediately fixed and stained for E-cadherin and actin. The fluorescence images show that the actin cytoskeleton remained intact, and that the fine spatial organization of E-cadherin at the cell–cell contact was the same as that before photobleaching. We also examined whether photobleaching of EcadGFP was reversible. Fig. 8 B shows the effects on EcadGFP after 1/2 of a cell was photobleached. The EcadGFP fluorescence was monitored in the photobleached half of the cell (Fig. 8 B, blue), the nonphotobleached half of the cell (Fig. 8 B, red), and the entire cell (Fig. 8 B, green). These data show that the photobleached part of the cell recovered EcadGFP fluorescence while, at the same rate, the nonphotobleached part of the cell lost EcadGFP fluorescence. The average intensity of EcadGFP fluorescence remained constant throughout the entire cell, reflecting the fact that the EcadGFP fluorescence was irreversibly photobleached. These results also show that the entire pool of EcadGFP in the cell was mobile and exchanged within 45 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)

Photobleach-recovery analysis shows a highly  mobile pool of EcadGFP coalesces into immobile puncta.  A shows a live cell before  and after photobleaching.  The box indicates where the  cell was photobleached. The  arrow points to an area that  formed a contact during the  photobleach. The cells were  fixed in formaldehyde and  stained with phalloidin and  mAb 3G8. B shows the fluorescence recovery curves of a  single noncontacting cell in  which half of the cell was  photobleached (blue). EcadGFP fluorescence of the nonbleached region (red) and  the entire cell (green) was  monitored during recovery.  Notice that the EcadGFP fluorescence values equalize in  the photobleached and nonphotobleached areas. C shows  the first 3 min of photobleach-recovery data of noncontacting membrane regions photobleached with a  5.8-μm (pink) and 3-μm  (black) circle. The relative  fluorescence is scaled between the fluorescence intensity just after bleaching and  equilibrium. The lines show  the theoretical recovery  curves for each region with a  diffusion coefficient of 3 ×  10−10 cm2/s. Note that the  smaller photobleach circle (black line) recovers more quickly. D shows images taken before, 0.1 min after, and 10 min after photobleaching a 5.8-μm-diameter circle in a region of membrane not involved in cell–cell contact (Membrane), a region of membrane in a  <15-min-old contact (New contact), a region of membrane in the middle of a <60-min-old contact (Puncta), and a membrane at the  edge of a >2-h-old contact (Plaque). For each experiment, 300 images were collected every 3.2 s at 0.11 μm/pixel. The circles mark the  photobleach region, and the colors correspond to the recovery curves shown in C (pink) and E. E shows photobleach-recovery data for  the bleached contact regions identified in D. The relative fluorescence is scaled to the pre-bleach intensity value. The mobile fraction  of EcadGFP in the new contact (blue), puncta (green), and plaque (orange) is 100, 50, and <10%, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Photobleach-recovery analysis shows a highly mobile pool of EcadGFP coalesces into immobile puncta. A shows a live cell before and after photobleaching. The box indicates where the cell was photobleached. The arrow points to an area that formed a contact during the photobleach. The cells were fixed in formaldehyde and stained with phalloidin and mAb 3G8. B shows the fluorescence recovery curves of a single noncontacting cell in which half of the cell was photobleached (blue). EcadGFP fluorescence of the nonbleached region (red) and the entire cell (green) was monitored during recovery. Notice that the EcadGFP fluorescence values equalize in the photobleached and nonphotobleached areas. C shows the first 3 min of photobleach-recovery data of noncontacting membrane regions photobleached with a 5.8-μm (pink) and 3-μm (black) circle. The relative fluorescence is scaled between the fluorescence intensity just after bleaching and equilibrium. The lines show the theoretical recovery curves for each region with a diffusion coefficient of 3 × 10−10 cm2/s. Note that the smaller photobleach circle (black line) recovers more quickly. D shows images taken before, 0.1 min after, and 10 min after photobleaching a 5.8-μm-diameter circle in a region of membrane not involved in cell–cell contact (Membrane), a region of membrane in a <15-min-old contact (New contact), a region of membrane in the middle of a <60-min-old contact (Puncta), and a membrane at the edge of a >2-h-old contact (Plaque). For each experiment, 300 images were collected every 3.2 s at 0.11 μm/pixel. The circles mark the photobleach region, and the colors correspond to the recovery curves shown in C (pink) and E. E shows photobleach-recovery data for the bleached contact regions identified in D. The relative fluorescence is scaled to the pre-bleach intensity value. The mobile fraction of EcadGFP in the new contact (blue), puncta (green), and plaque (orange) is 100, 50, and <10%, respectively.
Mentions: To gain further insight into the assembly dynamics of E-cadherin puncta and plaques, we developed a photobleaching-recovery method to measure the diffusion coefficient, mobile fraction, and redistribution of EcadGFP during different stages of contact development (see Materials and Methods). Photobleaching of cell–cell contacts neither disrupts the organization of adhering plasma membranes, induces retraction of membranes, nor changes membrane movements or dynamics (Fig. 8 A). Fig. 8 A shows the DIC and EcadGFP images for a live cell before and after photobleaching. After photobleaching, the cells were immediately fixed and stained for E-cadherin and actin. The fluorescence images show that the actin cytoskeleton remained intact, and that the fine spatial organization of E-cadherin at the cell–cell contact was the same as that before photobleaching. We also examined whether photobleaching of EcadGFP was reversible. Fig. 8 B shows the effects on EcadGFP after 1/2 of a cell was photobleached. The EcadGFP fluorescence was monitored in the photobleached half of the cell (Fig. 8 B, blue), the nonphotobleached half of the cell (Fig. 8 B, red), and the entire cell (Fig. 8 B, green). These data show that the photobleached part of the cell recovered EcadGFP fluorescence while, at the same rate, the nonphotobleached part of the cell lost EcadGFP fluorescence. The average intensity of EcadGFP fluorescence remained constant throughout the entire cell, reflecting the fact that the EcadGFP fluorescence was irreversibly photobleached. These results also show that the entire pool of EcadGFP in the cell was mobile and exchanged within 45 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