Limits...
Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics.

Le TL, Yap AS, Stow JL - J. Cell Biol. (1999)

Bottom Line: The reformation of cell junctions after replacement of Ca2+ was then found to be inhibited when recycling of endocytosed E-cadherin was disrupted by bafilomycin treatment.The endocytosis and recycling of E-cadherin and of the transferrin receptor were similarly inhibited by potassium depletion and by bafilomycin treatment, and both proteins were accumulated in intracellular compartments by an 18 degrees C temperature block, suggesting that endocytosis may occur via a clathrin-mediated pathway.We conclude that a pool of surface E-cadherin is constantly trafficked through an endocytic, recycling pathway and that this may provide a mechanism for regulating the availability of E-cadherin for junction formation in development, tissue remodeling, and tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular and Cellular Biology, The University of Queensland, Brisbane, 4072 Queensland, Australia.

ABSTRACT
E-Cadherin plays critical roles in many aspects of cell adhesion, epithelial development, and the establishment and maintenance of epithelial polarity. The fate of E-cadherin once it is delivered to the basolateral cell surface, and the mechanisms which govern its participation in adherens junctions, are not well understood. Using surface biotinylation and recycling assays, we observed that some of the cell surface E-cadherin is actively internalized and is then recycled back to the plasma membrane. The pool of E-cadherin undergoing endocytosis and recycling was markedly increased in cells without stable cell-cell contacts, i.e., in preconfluent cells and after cell contacts were disrupted by depletion of extracellular Ca2+, suggesting that endocytic trafficking of E-cadherin is regulated by cell-cell contact. The reformation of cell junctions after replacement of Ca2+ was then found to be inhibited when recycling of endocytosed E-cadherin was disrupted by bafilomycin treatment. The endocytosis and recycling of E-cadherin and of the transferrin receptor were similarly inhibited by potassium depletion and by bafilomycin treatment, and both proteins were accumulated in intracellular compartments by an 18 degrees C temperature block, suggesting that endocytosis may occur via a clathrin-mediated pathway. We conclude that a pool of surface E-cadherin is constantly trafficked through an endocytic, recycling pathway and that this may provide a mechanism for regulating the availability of E-cadherin for junction formation in development, tissue remodeling, and tumorigenesis.

Show MeSH

Related in: MedlinePlus

Endocytosis of E-cadherin after depletion of extracellular Ca2+. Internalization of E-cadherin was compared in untreated confluent MDCK cells and in cells incubated in medium containing EDTA to chelate extracellular Ca2+. (a) Immunofluorescence localization of E-cadherin in control MDCK cells (−EDTA) and MDCK cells incubated in DMEM containing 2.5 mM EDTA (+EDTA) and cycloheximide (10 μM) for 30 min. In the presence of EDTA cells show prominent intracellular vesicular staining of E-cadherin. (b) Quantification of relative fluorescence intensities at the cell surface and inside cells measured by SOM software showed that chelation of Ca2+ resulted in a dramatic increase in intracellular staining accompanied by a concomitant decrease in plasma membrane staining, indicative of E-cadherin endocytosis stimulated by EDTA. Data are means ± SEM . (c) Surface biotinylation. Cells were surface-biotinylated at 0°C (lane 1) and then incubated at 37°C in normal medium (lanes 3 and 5) or in medium containing EDTA (lanes 4 and 6) for 30 min to allow for internalization. Total biotinylated E-cadherin was unchanged in the total cell extracts under both these conditions (lanes 3 and 4). After glutathione stripping there was a significantly increased pool of internalized biotinylated E-cadherin after Ca2+ depletion (lane 6) compared to control cells (lane 5). Thus, EDTA treatment increased internalization of surface-biotinylated E-cadherin. (d) Surface trypsinization. Cells were incubated in normal media or in medium containing 2.5 mM EDTA for 30 min. Cell surface proteins were removed by trypsinization and the remaining E-cadherin in cell extracts was analyzed by SDS-PAGE and immunoblotting with a NH2 terminus antibody (3B8). Total cellular E-cadherin remained unchanged in the absence (lane 1) or presence (lane 2) of Ca2+ chelation. A small pool of internalized E-cadherin was detected after trypsin treatment in cells incubated in normal media (lane 3), but this pool was dramatically increased in the presence of EDTA (lane 4), showing increased internalization of surface E-cadherin. Results shown are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199726&req=5

Figure 7: Endocytosis of E-cadherin after depletion of extracellular Ca2+. Internalization of E-cadherin was compared in untreated confluent MDCK cells and in cells incubated in medium containing EDTA to chelate extracellular Ca2+. (a) Immunofluorescence localization of E-cadherin in control MDCK cells (−EDTA) and MDCK cells incubated in DMEM containing 2.5 mM EDTA (+EDTA) and cycloheximide (10 μM) for 30 min. In the presence of EDTA cells show prominent intracellular vesicular staining of E-cadherin. (b) Quantification of relative fluorescence intensities at the cell surface and inside cells measured by SOM software showed that chelation of Ca2+ resulted in a dramatic increase in intracellular staining accompanied by a concomitant decrease in plasma membrane staining, indicative of E-cadherin endocytosis stimulated by EDTA. Data are means ± SEM . (c) Surface biotinylation. Cells were surface-biotinylated at 0°C (lane 1) and then incubated at 37°C in normal medium (lanes 3 and 5) or in medium containing EDTA (lanes 4 and 6) for 30 min to allow for internalization. Total biotinylated E-cadherin was unchanged in the total cell extracts under both these conditions (lanes 3 and 4). After glutathione stripping there was a significantly increased pool of internalized biotinylated E-cadherin after Ca2+ depletion (lane 6) compared to control cells (lane 5). Thus, EDTA treatment increased internalization of surface-biotinylated E-cadherin. (d) Surface trypsinization. Cells were incubated in normal media or in medium containing 2.5 mM EDTA for 30 min. Cell surface proteins were removed by trypsinization and the remaining E-cadherin in cell extracts was analyzed by SDS-PAGE and immunoblotting with a NH2 terminus antibody (3B8). Total cellular E-cadherin remained unchanged in the absence (lane 1) or presence (lane 2) of Ca2+ chelation. A small pool of internalized E-cadherin was detected after trypsin treatment in cells incubated in normal media (lane 3), but this pool was dramatically increased in the presence of EDTA (lane 4), showing increased internalization of surface E-cadherin. Results shown are representative of three independent experiments.

Mentions: To further investigate the influence of cell-cell contact on E-cadherin recycling, we examined the effect of EDTA on epithelial morphology and E-cadherin localization in confluent MDCK monolayers. Chelation of extracellular Ca2+ disrupts epithelial cohesion, at least partly through inhibition of the adhesive binding activity of the E-cadherin ectodomain (Takeichi et al. 1981; Kemler et al. 1989; Kartenbeck et al. 1991). As shown in Fig. 6, exposure of MDCK cells to EDTA (2.5 mM) induces a rapid and reversible change in monolayer organization. Within 10–15 min cells began to retract from one another, and by 45 min there were mostly isolated cells lacking cell-cell contacts (Fig. 6 b). Restoration of extracellular Ca2+ rapidly restored cell-cell contacts, and by 1 h most cells had spread to reform extensive regions of confluence (Fig. 6 d). Staining for E-cadherin showed that EDTA induced the rapid internalization of surface E-cadherins with intense intracellular punctate labeling apparent after Ca2+ chelation (Fig. 7 a and Fig. 8 b) compared to the predominant cell surface staining of E-cadherin in contact zones in untreated cells (Fig. 7 a and Fig. 8 a). Quantitative immunofluorescence analysis indicated that whereas in control cells the majority of E-cadherin staining was at the cell surface, after treatment with EDTA E-cadherin was now predominantly intracellular (Fig. 7 b). Upon restoration of extracellular Ca2+, E-cadherin staining reappeared at sites of cell-cell contact, with a concomitant reduction in intracellular staining (Fig. 8 d).


Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics.

Le TL, Yap AS, Stow JL - J. Cell Biol. (1999)

Endocytosis of E-cadherin after depletion of extracellular Ca2+. Internalization of E-cadherin was compared in untreated confluent MDCK cells and in cells incubated in medium containing EDTA to chelate extracellular Ca2+. (a) Immunofluorescence localization of E-cadherin in control MDCK cells (−EDTA) and MDCK cells incubated in DMEM containing 2.5 mM EDTA (+EDTA) and cycloheximide (10 μM) for 30 min. In the presence of EDTA cells show prominent intracellular vesicular staining of E-cadherin. (b) Quantification of relative fluorescence intensities at the cell surface and inside cells measured by SOM software showed that chelation of Ca2+ resulted in a dramatic increase in intracellular staining accompanied by a concomitant decrease in plasma membrane staining, indicative of E-cadherin endocytosis stimulated by EDTA. Data are means ± SEM . (c) Surface biotinylation. Cells were surface-biotinylated at 0°C (lane 1) and then incubated at 37°C in normal medium (lanes 3 and 5) or in medium containing EDTA (lanes 4 and 6) for 30 min to allow for internalization. Total biotinylated E-cadherin was unchanged in the total cell extracts under both these conditions (lanes 3 and 4). After glutathione stripping there was a significantly increased pool of internalized biotinylated E-cadherin after Ca2+ depletion (lane 6) compared to control cells (lane 5). Thus, EDTA treatment increased internalization of surface-biotinylated E-cadherin. (d) Surface trypsinization. Cells were incubated in normal media or in medium containing 2.5 mM EDTA for 30 min. Cell surface proteins were removed by trypsinization and the remaining E-cadherin in cell extracts was analyzed by SDS-PAGE and immunoblotting with a NH2 terminus antibody (3B8). Total cellular E-cadherin remained unchanged in the absence (lane 1) or presence (lane 2) of Ca2+ chelation. A small pool of internalized E-cadherin was detected after trypsin treatment in cells incubated in normal media (lane 3), but this pool was dramatically increased in the presence of EDTA (lane 4), showing increased internalization of surface E-cadherin. Results shown are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199726&req=5

Figure 7: Endocytosis of E-cadherin after depletion of extracellular Ca2+. Internalization of E-cadherin was compared in untreated confluent MDCK cells and in cells incubated in medium containing EDTA to chelate extracellular Ca2+. (a) Immunofluorescence localization of E-cadherin in control MDCK cells (−EDTA) and MDCK cells incubated in DMEM containing 2.5 mM EDTA (+EDTA) and cycloheximide (10 μM) for 30 min. In the presence of EDTA cells show prominent intracellular vesicular staining of E-cadherin. (b) Quantification of relative fluorescence intensities at the cell surface and inside cells measured by SOM software showed that chelation of Ca2+ resulted in a dramatic increase in intracellular staining accompanied by a concomitant decrease in plasma membrane staining, indicative of E-cadherin endocytosis stimulated by EDTA. Data are means ± SEM . (c) Surface biotinylation. Cells were surface-biotinylated at 0°C (lane 1) and then incubated at 37°C in normal medium (lanes 3 and 5) or in medium containing EDTA (lanes 4 and 6) for 30 min to allow for internalization. Total biotinylated E-cadherin was unchanged in the total cell extracts under both these conditions (lanes 3 and 4). After glutathione stripping there was a significantly increased pool of internalized biotinylated E-cadherin after Ca2+ depletion (lane 6) compared to control cells (lane 5). Thus, EDTA treatment increased internalization of surface-biotinylated E-cadherin. (d) Surface trypsinization. Cells were incubated in normal media or in medium containing 2.5 mM EDTA for 30 min. Cell surface proteins were removed by trypsinization and the remaining E-cadherin in cell extracts was analyzed by SDS-PAGE and immunoblotting with a NH2 terminus antibody (3B8). Total cellular E-cadherin remained unchanged in the absence (lane 1) or presence (lane 2) of Ca2+ chelation. A small pool of internalized E-cadherin was detected after trypsin treatment in cells incubated in normal media (lane 3), but this pool was dramatically increased in the presence of EDTA (lane 4), showing increased internalization of surface E-cadherin. Results shown are representative of three independent experiments.
Mentions: To further investigate the influence of cell-cell contact on E-cadherin recycling, we examined the effect of EDTA on epithelial morphology and E-cadherin localization in confluent MDCK monolayers. Chelation of extracellular Ca2+ disrupts epithelial cohesion, at least partly through inhibition of the adhesive binding activity of the E-cadherin ectodomain (Takeichi et al. 1981; Kemler et al. 1989; Kartenbeck et al. 1991). As shown in Fig. 6, exposure of MDCK cells to EDTA (2.5 mM) induces a rapid and reversible change in monolayer organization. Within 10–15 min cells began to retract from one another, and by 45 min there were mostly isolated cells lacking cell-cell contacts (Fig. 6 b). Restoration of extracellular Ca2+ rapidly restored cell-cell contacts, and by 1 h most cells had spread to reform extensive regions of confluence (Fig. 6 d). Staining for E-cadherin showed that EDTA induced the rapid internalization of surface E-cadherins with intense intracellular punctate labeling apparent after Ca2+ chelation (Fig. 7 a and Fig. 8 b) compared to the predominant cell surface staining of E-cadherin in contact zones in untreated cells (Fig. 7 a and Fig. 8 a). Quantitative immunofluorescence analysis indicated that whereas in control cells the majority of E-cadherin staining was at the cell surface, after treatment with EDTA E-cadherin was now predominantly intracellular (Fig. 7 b). Upon restoration of extracellular Ca2+, E-cadherin staining reappeared at sites of cell-cell contact, with a concomitant reduction in intracellular staining (Fig. 8 d).

Bottom Line: The reformation of cell junctions after replacement of Ca2+ was then found to be inhibited when recycling of endocytosed E-cadherin was disrupted by bafilomycin treatment.The endocytosis and recycling of E-cadherin and of the transferrin receptor were similarly inhibited by potassium depletion and by bafilomycin treatment, and both proteins were accumulated in intracellular compartments by an 18 degrees C temperature block, suggesting that endocytosis may occur via a clathrin-mediated pathway.We conclude that a pool of surface E-cadherin is constantly trafficked through an endocytic, recycling pathway and that this may provide a mechanism for regulating the availability of E-cadherin for junction formation in development, tissue remodeling, and tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular and Cellular Biology, The University of Queensland, Brisbane, 4072 Queensland, Australia.

ABSTRACT
E-Cadherin plays critical roles in many aspects of cell adhesion, epithelial development, and the establishment and maintenance of epithelial polarity. The fate of E-cadherin once it is delivered to the basolateral cell surface, and the mechanisms which govern its participation in adherens junctions, are not well understood. Using surface biotinylation and recycling assays, we observed that some of the cell surface E-cadherin is actively internalized and is then recycled back to the plasma membrane. The pool of E-cadherin undergoing endocytosis and recycling was markedly increased in cells without stable cell-cell contacts, i.e., in preconfluent cells and after cell contacts were disrupted by depletion of extracellular Ca2+, suggesting that endocytic trafficking of E-cadherin is regulated by cell-cell contact. The reformation of cell junctions after replacement of Ca2+ was then found to be inhibited when recycling of endocytosed E-cadherin was disrupted by bafilomycin treatment. The endocytosis and recycling of E-cadherin and of the transferrin receptor were similarly inhibited by potassium depletion and by bafilomycin treatment, and both proteins were accumulated in intracellular compartments by an 18 degrees C temperature block, suggesting that endocytosis may occur via a clathrin-mediated pathway. We conclude that a pool of surface E-cadherin is constantly trafficked through an endocytic, recycling pathway and that this may provide a mechanism for regulating the availability of E-cadherin for junction formation in development, tissue remodeling, and tumorigenesis.

Show MeSH
Related in: MedlinePlus