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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.

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Endocytosis of E-cadherin is inhibited by K+ depletion. (a) Control cells (lane 1), cells pretreated with hypotonic shock alone (lane 2), or cells which were pretreated with hypotonic shock for 5 min then also incubated in K+-free medium for 15 min (lane 3) were surface-biotinylated. Biotinylated proteins were collected and analyzed by SDS-PAGE and immunoblotted to detect E-cadherin, transferrin receptor, and Na+K+ATPase. A set of duplicate cells was then treated and surface-biotinylated and then incubated at 37°C in either K+-free medium (lanes 6 and 7) or normal medium (lane 5) to allow for internalization of surface proteins. Cells were glutathione-stripped and the internal pool recovered with streptavidin beads. Biotinylated E-cadherin and TfR were both internalized in control cells (lane 5) and in cells treated with hypotonic shock alone (lane 6), but internalization of both proteins was effectively blocked in cells depleted of K+ using hypotonic shock and K+-free medium (lane 7). Na+K+ATPase did not internalize under either control or K+ depletion conditions (lanes 5–7). The results are representative of four separate experiments. (b) Clathrin-independent endocytosis of FITC-ricin. Cells were incubated in either normal media or were hypotonically shocked and incubated in K+-free media as outlined above. Cells were then surface-labeled with FITC-ricin at 4°C for 1 h and then incubated in normal or K+-free media for 30 min at 37°C to allow for internalization. Nonendocytosed FITC-ricin was removed by several washes with 0.2 M lactose in PBS. Cells were then fixed and the amount of internalized FITC-ricin was viewed and analyzed by confocal microscopy using SOM software. Similar amounts of intracellular FITC-ricin staining were obtained in both sets of cells. Inhibition of clathrin-mediated endocytosis by K+ depletion under the shown conditions in a and b, did not affect FITC-ricin uptake. Data are means ± SEM .
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Figure 9: Endocytosis of E-cadherin is inhibited by K+ depletion. (a) Control cells (lane 1), cells pretreated with hypotonic shock alone (lane 2), or cells which were pretreated with hypotonic shock for 5 min then also incubated in K+-free medium for 15 min (lane 3) were surface-biotinylated. Biotinylated proteins were collected and analyzed by SDS-PAGE and immunoblotted to detect E-cadherin, transferrin receptor, and Na+K+ATPase. A set of duplicate cells was then treated and surface-biotinylated and then incubated at 37°C in either K+-free medium (lanes 6 and 7) or normal medium (lane 5) to allow for internalization of surface proteins. Cells were glutathione-stripped and the internal pool recovered with streptavidin beads. Biotinylated E-cadherin and TfR were both internalized in control cells (lane 5) and in cells treated with hypotonic shock alone (lane 6), but internalization of both proteins was effectively blocked in cells depleted of K+ using hypotonic shock and K+-free medium (lane 7). Na+K+ATPase did not internalize under either control or K+ depletion conditions (lanes 5–7). The results are representative of four separate experiments. (b) Clathrin-independent endocytosis of FITC-ricin. Cells were incubated in either normal media or were hypotonically shocked and incubated in K+-free media as outlined above. Cells were then surface-labeled with FITC-ricin at 4°C for 1 h and then incubated in normal or K+-free media for 30 min at 37°C to allow for internalization. Nonendocytosed FITC-ricin was removed by several washes with 0.2 M lactose in PBS. Cells were then fixed and the amount of internalized FITC-ricin was viewed and analyzed by confocal microscopy using SOM software. Similar amounts of intracellular FITC-ricin staining were obtained in both sets of cells. Inhibition of clathrin-mediated endocytosis by K+ depletion under the shown conditions in a and b, did not affect FITC-ricin uptake. Data are means ± SEM .

Mentions: Endocytosis of E-cadherin could occur through either clathrin-mediated or clathrin-independent pathways. To study whether E-cadherin is internalized via clathrin-dependent endocytosis, we used K+ depletion combined with hypotonic shock, a maneuver which has been shown to specifically inhibit clathrin-coated pit uptake of the low density lipoprotein receptor (Larkin et al. 1983) and other receptors. MDCK cells were preincubated with K+-free media, then gently rinsed and exposed to hypotonic K+-free media followed by incubation in K+-free media. Cells were then surface-biotinylated, incubated at 37°C for internalization, and then the surface was glutathione stripped. In control cells some of the surface-biotinylated E-cadherin was recovered in an internal pool (Fig. 9 a, lane 5; see also Fig. 2 a). A similar proportion of E-cadherin was internalized after hypotonic shock alone (Fig. 9 a, lane 6) which did not arrest E-cadherin internalization. However in cells treated with hypotonic shock followed by incubation in K+-free media there was no biotinylated E-cadherin internalized (Fig. 9 a, lane 7). Under the same conditions, the internalization of biotinylated TfR, which is known to be taken up by a clathrin-dependent pathway, was also blocked (Fig. 9 a, lane 7), whereas the surface residence of Na+K+ATPase, which in our hands is not internalized, was unchanged. In contrast, the uptake of FITC-labeled ricin, which occurs via a clathrin-independent mechanism (for review see Sandvig and van Deurs 1996), was unaffected by hypotonic shock and K+ depletion (Fig. 9 b). The same levels of FITC-ricin staining were measured in treated and untreated monolayers. Thus, K+ depletion effectively and specifically blocked clathrin-dependent uptake, implicating such a pathway in the internalization of E-cadherin.


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 is inhibited by K+ depletion. (a) Control cells (lane 1), cells pretreated with hypotonic shock alone (lane 2), or cells which were pretreated with hypotonic shock for 5 min then also incubated in K+-free medium for 15 min (lane 3) were surface-biotinylated. Biotinylated proteins were collected and analyzed by SDS-PAGE and immunoblotted to detect E-cadherin, transferrin receptor, and Na+K+ATPase. A set of duplicate cells was then treated and surface-biotinylated and then incubated at 37°C in either K+-free medium (lanes 6 and 7) or normal medium (lane 5) to allow for internalization of surface proteins. Cells were glutathione-stripped and the internal pool recovered with streptavidin beads. Biotinylated E-cadherin and TfR were both internalized in control cells (lane 5) and in cells treated with hypotonic shock alone (lane 6), but internalization of both proteins was effectively blocked in cells depleted of K+ using hypotonic shock and K+-free medium (lane 7). Na+K+ATPase did not internalize under either control or K+ depletion conditions (lanes 5–7). The results are representative of four separate experiments. (b) Clathrin-independent endocytosis of FITC-ricin. Cells were incubated in either normal media or were hypotonically shocked and incubated in K+-free media as outlined above. Cells were then surface-labeled with FITC-ricin at 4°C for 1 h and then incubated in normal or K+-free media for 30 min at 37°C to allow for internalization. Nonendocytosed FITC-ricin was removed by several washes with 0.2 M lactose in PBS. Cells were then fixed and the amount of internalized FITC-ricin was viewed and analyzed by confocal microscopy using SOM software. Similar amounts of intracellular FITC-ricin staining were obtained in both sets of cells. Inhibition of clathrin-mediated endocytosis by K+ depletion under the shown conditions in a and b, did not affect FITC-ricin uptake. Data are means ± SEM .
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Figure 9: Endocytosis of E-cadherin is inhibited by K+ depletion. (a) Control cells (lane 1), cells pretreated with hypotonic shock alone (lane 2), or cells which were pretreated with hypotonic shock for 5 min then also incubated in K+-free medium for 15 min (lane 3) were surface-biotinylated. Biotinylated proteins were collected and analyzed by SDS-PAGE and immunoblotted to detect E-cadherin, transferrin receptor, and Na+K+ATPase. A set of duplicate cells was then treated and surface-biotinylated and then incubated at 37°C in either K+-free medium (lanes 6 and 7) or normal medium (lane 5) to allow for internalization of surface proteins. Cells were glutathione-stripped and the internal pool recovered with streptavidin beads. Biotinylated E-cadherin and TfR were both internalized in control cells (lane 5) and in cells treated with hypotonic shock alone (lane 6), but internalization of both proteins was effectively blocked in cells depleted of K+ using hypotonic shock and K+-free medium (lane 7). Na+K+ATPase did not internalize under either control or K+ depletion conditions (lanes 5–7). The results are representative of four separate experiments. (b) Clathrin-independent endocytosis of FITC-ricin. Cells were incubated in either normal media or were hypotonically shocked and incubated in K+-free media as outlined above. Cells were then surface-labeled with FITC-ricin at 4°C for 1 h and then incubated in normal or K+-free media for 30 min at 37°C to allow for internalization. Nonendocytosed FITC-ricin was removed by several washes with 0.2 M lactose in PBS. Cells were then fixed and the amount of internalized FITC-ricin was viewed and analyzed by confocal microscopy using SOM software. Similar amounts of intracellular FITC-ricin staining were obtained in both sets of cells. Inhibition of clathrin-mediated endocytosis by K+ depletion under the shown conditions in a and b, did not affect FITC-ricin uptake. Data are means ± SEM .
Mentions: Endocytosis of E-cadherin could occur through either clathrin-mediated or clathrin-independent pathways. To study whether E-cadherin is internalized via clathrin-dependent endocytosis, we used K+ depletion combined with hypotonic shock, a maneuver which has been shown to specifically inhibit clathrin-coated pit uptake of the low density lipoprotein receptor (Larkin et al. 1983) and other receptors. MDCK cells were preincubated with K+-free media, then gently rinsed and exposed to hypotonic K+-free media followed by incubation in K+-free media. Cells were then surface-biotinylated, incubated at 37°C for internalization, and then the surface was glutathione stripped. In control cells some of the surface-biotinylated E-cadherin was recovered in an internal pool (Fig. 9 a, lane 5; see also Fig. 2 a). A similar proportion of E-cadherin was internalized after hypotonic shock alone (Fig. 9 a, lane 6) which did not arrest E-cadherin internalization. However in cells treated with hypotonic shock followed by incubation in K+-free media there was no biotinylated E-cadherin internalized (Fig. 9 a, lane 7). Under the same conditions, the internalization of biotinylated TfR, which is known to be taken up by a clathrin-dependent pathway, was also blocked (Fig. 9 a, lane 7), whereas the surface residence of Na+K+ATPase, which in our hands is not internalized, was unchanged. In contrast, the uptake of FITC-labeled ricin, which occurs via a clathrin-independent mechanism (for review see Sandvig and van Deurs 1996), was unaffected by hypotonic shock and K+ depletion (Fig. 9 b). The same levels of FITC-ricin staining were measured in treated and untreated monolayers. Thus, K+ depletion effectively and specifically blocked clathrin-dependent uptake, implicating such a pathway in the internalization of E-cadherin.

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