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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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Simultaneous internalization of PLAP and gp114. Electron micrographs of MDCK cells after cross-linking and internalization with a mixture of polyclonal PLAP antibodies and monoclonal gp114 antibodies. The respective antibodies were detected with 12-nm gold coupled anti-rabbit antibodies and 6-nm gold coupled anti-mouse antibodies. Caveolar-like structures are double labeled at the apical plasma membrane (A an B). Not all structures are double labeled as shown in C where one endocytic structure is double labeled (arrow), while others contain one type of gold label. Basolateral caveolar-like structures are also double labeled (D, arrowhead). Bar: (A and C) 150 nm; (B and D) 100 nm.
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Figure 9: Simultaneous internalization of PLAP and gp114. Electron micrographs of MDCK cells after cross-linking and internalization with a mixture of polyclonal PLAP antibodies and monoclonal gp114 antibodies. The respective antibodies were detected with 12-nm gold coupled anti-rabbit antibodies and 6-nm gold coupled anti-mouse antibodies. Caveolar-like structures are double labeled at the apical plasma membrane (A an B). Not all structures are double labeled as shown in C where one endocytic structure is double labeled (arrow), while others contain one type of gold label. Basolateral caveolar-like structures are also double labeled (D, arrowhead). Bar: (A and C) 150 nm; (B and D) 100 nm.

Mentions: These ultrastructural data together with the internalization rates showed that the clathrin-dependent uptake of LDLR-CT22 is clearly different from the endocytosis of PLAP and gp114, the latter of which appear to employ the same internalization mechanism. This was confirmed by simultaneous internalization of PLAP and gp114 labeled with rabbit and mouse antibodies, respectively. The majority of the structures was labeled with both markers (Fig. 9). Cross-linking and internalization of PLAP or gp114 together with HA in influenza virus-infected cells also resulted in colabeling of caveolar-like and endocytic structures (data not shown). This shows that clustered raft proteins employ the same structures for internalization from the apical membrane. When gp114 and LDLR-CT22 were simultaneously internalized they were detected in different structures (Fig. 10 A). Although the internalization of clustered raft proteins on the one hand and LDLR-CT22 on the other hand use different mechanisms, they do enter the same endosome structures (Fig. 10 B). It is important to note that we never observed LDLR-CT22 in basolateral caveolae.


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)

Simultaneous internalization of PLAP and gp114. Electron micrographs of MDCK cells after cross-linking and internalization with a mixture of polyclonal PLAP antibodies and monoclonal gp114 antibodies. The respective antibodies were detected with 12-nm gold coupled anti-rabbit antibodies and 6-nm gold coupled anti-mouse antibodies. Caveolar-like structures are double labeled at the apical plasma membrane (A an B). Not all structures are double labeled as shown in C where one endocytic structure is double labeled (arrow), while others contain one type of gold label. Basolateral caveolar-like structures are also double labeled (D, arrowhead). Bar: (A and C) 150 nm; (B and D) 100 nm.
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Related In: Results  -  Collection

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Figure 9: Simultaneous internalization of PLAP and gp114. Electron micrographs of MDCK cells after cross-linking and internalization with a mixture of polyclonal PLAP antibodies and monoclonal gp114 antibodies. The respective antibodies were detected with 12-nm gold coupled anti-rabbit antibodies and 6-nm gold coupled anti-mouse antibodies. Caveolar-like structures are double labeled at the apical plasma membrane (A an B). Not all structures are double labeled as shown in C where one endocytic structure is double labeled (arrow), while others contain one type of gold label. Basolateral caveolar-like structures are also double labeled (D, arrowhead). Bar: (A and C) 150 nm; (B and D) 100 nm.
Mentions: These ultrastructural data together with the internalization rates showed that the clathrin-dependent uptake of LDLR-CT22 is clearly different from the endocytosis of PLAP and gp114, the latter of which appear to employ the same internalization mechanism. This was confirmed by simultaneous internalization of PLAP and gp114 labeled with rabbit and mouse antibodies, respectively. The majority of the structures was labeled with both markers (Fig. 9). Cross-linking and internalization of PLAP or gp114 together with HA in influenza virus-infected cells also resulted in colabeling of caveolar-like and endocytic structures (data not shown). This shows that clustered raft proteins employ the same structures for internalization from the apical membrane. When gp114 and LDLR-CT22 were simultaneously internalized they were detected in different structures (Fig. 10 A). Although the internalization of clustered raft proteins on the one hand and LDLR-CT22 on the other hand use different mechanisms, they do enter the same endosome structures (Fig. 10 B). It is important to note that we never observed LDLR-CT22 in basolateral caveolae.

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