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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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Cholesterol dependence of the apical internalization of PLAP and LDLR-CT22. Internalization of cross-linked PLAP and LDLR-CT22 from the apical plasma membrane at 37°C measured by 125I-labeled secondary antibodies. Internalization is scaled to 100% for the maximum internalization.
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Figure 12: Cholesterol dependence of the apical internalization of PLAP and LDLR-CT22. Internalization of cross-linked PLAP and LDLR-CT22 from the apical plasma membrane at 37°C measured by 125I-labeled secondary antibodies. Internalization is scaled to 100% for the maximum internalization.

Mentions: To further investigate the properties of the apical caveolar-like structures two additional features of caveolae, namely the presence of caveolin-1 and the dependence on cholesterol, were investigated. In previous experiments, we have detected caveolin-1 by a commercial polyclonal antibody N20 (Scheiffele et al. 1998) and did not detect any morphologically apparent caveolae at the apical membrane. Here we used the same antibody and found an accumulation of caveolin-1 in the clusters induced by PLAP cross-linking at 4–8°C (Fig. 3). When the cells were brought to 37°C and the clusters were internalized, the apical caveolar-like structures also labeled for caveolin-1 (Fig. 11 A). The PLAP-labeled, caveolar-like structures on the basolateral membrane were also identified as caveolae by their labeling with caveolin-1 (Fig. 11 B). The same results were obtained for gp114 (data not shown). Cholesterol removal reduced the uptake of PLAP to background levels (Fig. 12). If the maximum internalization was scaled to 100%, this corresponds to a reduction to <5%. The same results were obtained for gp114 (data not shown). Under the same conditions of CD treatment the internalization of LDLR-CT22 was reduced by ∼50% after the cholesterol depletion (Fig. 12). Electron microscopical analysis showed that after CD treatment no caveolar-like structures were present and no gold associated to PLAP or gp114 had been internalized (data not shown). Tight junctions were intact but basolateral caveolae had disappeared in accordance with previous results (Hailstones et al. 1998). Clathrin-coated pits were still present at the apical and basolateral plasma membrane, and LDLR was still internalized via clathrin-coated pits in LDLR-CT22–expressing cells (data not shown).


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)

Cholesterol dependence of the apical internalization of PLAP and LDLR-CT22. Internalization of cross-linked PLAP and LDLR-CT22 from the apical plasma membrane at 37°C measured by 125I-labeled secondary antibodies. Internalization is scaled to 100% for the maximum internalization.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169379&req=5

Figure 12: Cholesterol dependence of the apical internalization of PLAP and LDLR-CT22. Internalization of cross-linked PLAP and LDLR-CT22 from the apical plasma membrane at 37°C measured by 125I-labeled secondary antibodies. Internalization is scaled to 100% for the maximum internalization.
Mentions: To further investigate the properties of the apical caveolar-like structures two additional features of caveolae, namely the presence of caveolin-1 and the dependence on cholesterol, were investigated. In previous experiments, we have detected caveolin-1 by a commercial polyclonal antibody N20 (Scheiffele et al. 1998) and did not detect any morphologically apparent caveolae at the apical membrane. Here we used the same antibody and found an accumulation of caveolin-1 in the clusters induced by PLAP cross-linking at 4–8°C (Fig. 3). When the cells were brought to 37°C and the clusters were internalized, the apical caveolar-like structures also labeled for caveolin-1 (Fig. 11 A). The PLAP-labeled, caveolar-like structures on the basolateral membrane were also identified as caveolae by their labeling with caveolin-1 (Fig. 11 B). The same results were obtained for gp114 (data not shown). Cholesterol removal reduced the uptake of PLAP to background levels (Fig. 12). If the maximum internalization was scaled to 100%, this corresponds to a reduction to <5%. The same results were obtained for gp114 (data not shown). Under the same conditions of CD treatment the internalization of LDLR-CT22 was reduced by ∼50% after the cholesterol depletion (Fig. 12). Electron microscopical analysis showed that after CD treatment no caveolar-like structures were present and no gold associated to PLAP or gp114 had been internalized (data not shown). Tight junctions were intact but basolateral caveolae had disappeared in accordance with previous results (Hailstones et al. 1998). Clathrin-coated pits were still present at the apical and basolateral plasma membrane, and LDLR was still internalized via clathrin-coated pits in LDLR-CT22–expressing cells (data not shown).

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