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Induction of caveolae in the apical plasma membrane of Madin-Darby canine kidney cells.

Verkade P, Harder T, Lafont F, Simons K - J. Cell Biol. (2000)

Bottom Line: In this paper, we have analyzed the behavior of antibody cross-linked raft-associated proteins on the surface of MDCK cells.We observed that cross-linking of membrane proteins gave different results depending on whether cross-linking occurred on the apical or basolateral plasma membrane.Since caveolae are normally present on the basolateral membrane but lacking from the apical side, our data demonstrate that antibody cross-linking induced the formation of caveolae, which slowly internalized cross-linked clusters of raft-associated proteins.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Cell Biology and Biophysics Programme, D-69117 Heidelberg, Germany.

ABSTRACT
In this paper, we have analyzed the behavior of antibody cross-linked raft-associated proteins on the surface of MDCK cells. We observed that cross-linking of membrane proteins gave different results depending on whether cross-linking occurred on the apical or basolateral plasma membrane. Whereas antibody cross-linking induced the formation of large clusters on the basolateral membrane, resembling those observed on the surface of fibroblasts (Harder, T., P. Scheiffele, P. Verkade, and K. Simons. 1998. J. Cell Biol. 929-942), only small ( approximately 100 nm) clusters formed on the apical plasma membrane. Cross-linked apical raft proteins e.g., GPI-anchored placental alkaline phosphatase (PLAP), influenza hemagglutinin, and gp114 coclustered and were internalized slowly ( approximately 10% after 60 min). Endocytosis occurred through surface invaginations that corresponded in size to caveolae and were labeled with caveolin-1 antibodies. Upon cholesterol depletion the internalization of PLAP was completely inhibited. In contrast, when a non-raft protein, the mutant LDL receptor LDLR-CT22, was cross-linked, it was excluded from the clusters of raft proteins and was rapidly internalized via clathrin-coated pits. Since caveolae are normally present on the basolateral membrane but lacking from the apical side, our data demonstrate that antibody cross-linking induced the formation of caveolae, which slowly internalized cross-linked clusters of raft-associated proteins.

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Quantitation of PLAP and gp114 internalizing structures. Four types of morphologically distinct structures were quantitated at four time points. Apical caveolar-like structures (ACS): apical plasma membrane-associated structures with the characteristic 50–100-nm-round or flask-shaped morphological appearance of a single caveola or a bunch of grapes. Their width is not smaller than one-third of their length. Endocytic structures (ES): endosome-like structures appearing inside the cell. Apical plasma membrane-associated tubular structures (ATS): plasma membrane-associated tubular structures. They have a uniform form and their width is smaller than one-third of their length. Basolateral caveolar-like structures (BCS): Basolateral plasma membrane-associated structures with the characteristic 50–100-nm-round or flask-shaped morphological appearance of caveolae. Their width is not smaller than one-third of their length. The number of immunogold-labeled structures ± SEM per cell section at the given time points is given. The structures were identified on basis of their morphological criteria. 25 cell sections per time point were taken. Differences in the number of gold-labeled structures per time point were calculated with a One-way anova with three groups. In case of a significant difference, two adjacent time points were compared using a Student's t test or a Welch test in case variances between the groups were not equal. Differences between two time points are depicted with “=” if there was no difference, and with “<” if P < 0.05, indicating a significant difference.
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Figure 8: Quantitation of PLAP and gp114 internalizing structures. Four types of morphologically distinct structures were quantitated at four time points. Apical caveolar-like structures (ACS): apical plasma membrane-associated structures with the characteristic 50–100-nm-round or flask-shaped morphological appearance of a single caveola or a bunch of grapes. Their width is not smaller than one-third of their length. Endocytic structures (ES): endosome-like structures appearing inside the cell. Apical plasma membrane-associated tubular structures (ATS): plasma membrane-associated tubular structures. They have a uniform form and their width is smaller than one-third of their length. Basolateral caveolar-like structures (BCS): Basolateral plasma membrane-associated structures with the characteristic 50–100-nm-round or flask-shaped morphological appearance of caveolae. Their width is not smaller than one-third of their length. The number of immunogold-labeled structures ± SEM per cell section at the given time points is given. The structures were identified on basis of their morphological criteria. 25 cell sections per time point were taken. Differences in the number of gold-labeled structures per time point were calculated with a One-way anova with three groups. In case of a significant difference, two adjacent time points were compared using a Student's t test or a Welch test in case variances between the groups were not equal. Differences between two time points are depicted with “=” if there was no difference, and with “<” if P < 0.05, indicating a significant difference.

Mentions: Upon ultrastructural examination, we found that cross-linked PLAP was internalized via structures that had the morphological appearance of caveolae (Fig. 6A and Fig. B). Other gold-labeled structures associated with the apical plasma membrane had a tubular appearance (Fig. 6 C) and inside the cell, pleiomorphic endosome-like structures (Fig. 6D and Fig. E) were labeled. The Golgi apparatus (Fig. 6 D) and dense lysosomes were not labeled (not shown). A remarkable observation was that PLAP-associated gold label was also found in caveolar-like structures at the basolateral plasma membrane (Fig. 6F and Fig. G). Gp114 (Fig. 7) was found in the same types of structures as PLAP with the exception that gp114 was rarely found in tubular structures at the apical membrane. The time-dependent appearance of the different types of structures was quantitated (Fig. 8). At time 0 min of internalization, we did not detect any intracellular structures labeled, only the apical plasma membrane was covered with gold clusters. After 15 min of internalization of PLAP or gp114, apical caveolar-like invaginations and cytoplasmic endocytic structures were detected. Apical tubules or the basolateral caveolar-like structures could not be seen. The apical caveolar-like invaginations reached their maximum number after 15 min amounting to ∼1 caveolar profile per cell section. In comparison, we correspondingly found between 10 and 50 caveolar profiles on the basolateral side (unpublished data). The number of cytoplasmic endocytic structures kept increasing from 15 to 60 min. After 60 min of internalization of PLAP, apical tubules and basolateral caveolar-like structures were detected. The basolateral membrane itself was hardly labeled. This suggests that PLAP is recycled back to the apical membrane via tubules but is also transcytosed and found in basolateral caveolae. Gp114 was also found in basolateral caveolae. However, very few apical tubules were labeled, suggesting that there is little recycling of gp114.


Induction of caveolae in the apical plasma membrane of Madin-Darby canine kidney cells.

Verkade P, Harder T, Lafont F, Simons K - J. Cell Biol. (2000)

Quantitation of PLAP and gp114 internalizing structures. Four types of morphologically distinct structures were quantitated at four time points. Apical caveolar-like structures (ACS): apical plasma membrane-associated structures with the characteristic 50–100-nm-round or flask-shaped morphological appearance of a single caveola or a bunch of grapes. Their width is not smaller than one-third of their length. Endocytic structures (ES): endosome-like structures appearing inside the cell. Apical plasma membrane-associated tubular structures (ATS): plasma membrane-associated tubular structures. They have a uniform form and their width is smaller than one-third of their length. Basolateral caveolar-like structures (BCS): Basolateral plasma membrane-associated structures with the characteristic 50–100-nm-round or flask-shaped morphological appearance of caveolae. Their width is not smaller than one-third of their length. The number of immunogold-labeled structures ± SEM per cell section at the given time points is given. The structures were identified on basis of their morphological criteria. 25 cell sections per time point were taken. Differences in the number of gold-labeled structures per time point were calculated with a One-way anova with three groups. In case of a significant difference, two adjacent time points were compared using a Student's t test or a Welch test in case variances between the groups were not equal. Differences between two time points are depicted with “=” if there was no difference, and with “<” if P < 0.05, indicating a significant difference.
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Related In: Results  -  Collection

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Figure 8: Quantitation of PLAP and gp114 internalizing structures. Four types of morphologically distinct structures were quantitated at four time points. Apical caveolar-like structures (ACS): apical plasma membrane-associated structures with the characteristic 50–100-nm-round or flask-shaped morphological appearance of a single caveola or a bunch of grapes. Their width is not smaller than one-third of their length. Endocytic structures (ES): endosome-like structures appearing inside the cell. Apical plasma membrane-associated tubular structures (ATS): plasma membrane-associated tubular structures. They have a uniform form and their width is smaller than one-third of their length. Basolateral caveolar-like structures (BCS): Basolateral plasma membrane-associated structures with the characteristic 50–100-nm-round or flask-shaped morphological appearance of caveolae. Their width is not smaller than one-third of their length. The number of immunogold-labeled structures ± SEM per cell section at the given time points is given. The structures were identified on basis of their morphological criteria. 25 cell sections per time point were taken. Differences in the number of gold-labeled structures per time point were calculated with a One-way anova with three groups. In case of a significant difference, two adjacent time points were compared using a Student's t test or a Welch test in case variances between the groups were not equal. Differences between two time points are depicted with “=” if there was no difference, and with “<” if P < 0.05, indicating a significant difference.
Mentions: Upon ultrastructural examination, we found that cross-linked PLAP was internalized via structures that had the morphological appearance of caveolae (Fig. 6A and Fig. B). Other gold-labeled structures associated with the apical plasma membrane had a tubular appearance (Fig. 6 C) and inside the cell, pleiomorphic endosome-like structures (Fig. 6D and Fig. E) were labeled. The Golgi apparatus (Fig. 6 D) and dense lysosomes were not labeled (not shown). A remarkable observation was that PLAP-associated gold label was also found in caveolar-like structures at the basolateral plasma membrane (Fig. 6F and Fig. G). Gp114 (Fig. 7) was found in the same types of structures as PLAP with the exception that gp114 was rarely found in tubular structures at the apical membrane. The time-dependent appearance of the different types of structures was quantitated (Fig. 8). At time 0 min of internalization, we did not detect any intracellular structures labeled, only the apical plasma membrane was covered with gold clusters. After 15 min of internalization of PLAP or gp114, apical caveolar-like invaginations and cytoplasmic endocytic structures were detected. Apical tubules or the basolateral caveolar-like structures could not be seen. The apical caveolar-like invaginations reached their maximum number after 15 min amounting to ∼1 caveolar profile per cell section. In comparison, we correspondingly found between 10 and 50 caveolar profiles on the basolateral side (unpublished data). The number of cytoplasmic endocytic structures kept increasing from 15 to 60 min. After 60 min of internalization of PLAP, apical tubules and basolateral caveolar-like structures were detected. The basolateral membrane itself was hardly labeled. This suggests that PLAP is recycled back to the apical membrane via tubules but is also transcytosed and found in basolateral caveolae. Gp114 was also found in basolateral caveolae. However, very few apical tubules were labeled, suggesting that there is little recycling of gp114.

Bottom Line: In this paper, we have analyzed the behavior of antibody cross-linked raft-associated proteins on the surface of MDCK cells.We observed that cross-linking of membrane proteins gave different results depending on whether cross-linking occurred on the apical or basolateral plasma membrane.Since caveolae are normally present on the basolateral membrane but lacking from the apical side, our data demonstrate that antibody cross-linking induced the formation of caveolae, which slowly internalized cross-linked clusters of raft-associated proteins.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Cell Biology and Biophysics Programme, D-69117 Heidelberg, Germany.

ABSTRACT
In this paper, we have analyzed the behavior of antibody cross-linked raft-associated proteins on the surface of MDCK cells. We observed that cross-linking of membrane proteins gave different results depending on whether cross-linking occurred on the apical or basolateral plasma membrane. Whereas antibody cross-linking induced the formation of large clusters on the basolateral membrane, resembling those observed on the surface of fibroblasts (Harder, T., P. Scheiffele, P. Verkade, and K. Simons. 1998. J. Cell Biol. 929-942), only small ( approximately 100 nm) clusters formed on the apical plasma membrane. Cross-linked apical raft proteins e.g., GPI-anchored placental alkaline phosphatase (PLAP), influenza hemagglutinin, and gp114 coclustered and were internalized slowly ( approximately 10% after 60 min). Endocytosis occurred through surface invaginations that corresponded in size to caveolae and were labeled with caveolin-1 antibodies. Upon cholesterol depletion the internalization of PLAP was completely inhibited. In contrast, when a non-raft protein, the mutant LDL receptor LDLR-CT22, was cross-linked, it was excluded from the clusters of raft proteins and was rapidly internalized via clathrin-coated pits. Since caveolae are normally present on the basolateral membrane but lacking from the apical side, our data demonstrate that antibody cross-linking induced the formation of caveolae, which slowly internalized cross-linked clusters of raft-associated proteins.

Show MeSH
Related in: MedlinePlus