Limits...
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.

Show MeSH

Related in: MedlinePlus

PLAP and gp114 endocytosis from the apical membrane. Internalization of PLAP, gp114, and LDLR-CT22 from the apical plasma membrane at 37°C measured by 125I-labeled secondary antibodies (see also inset for a better view of PLAP and gp114). Numbers are expressed as percentage counts internalized compared with the total bound and internalized.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169379&req=5

Figure 5: PLAP and gp114 endocytosis from the apical membrane. Internalization of PLAP, gp114, and LDLR-CT22 from the apical plasma membrane at 37°C measured by 125I-labeled secondary antibodies (see also inset for a better view of PLAP and gp114). Numbers are expressed as percentage counts internalized compared with the total bound and internalized.

Mentions: Next we studied the internalization of cross-linked raft proteins from the apical plasma membrane. After cross-linking of the marker proteins at the apical surface and incubation at 37°C, we indeed observed that they were endocytosed. PLAP and gp114 were internalized slowly from the apical membrane (Fig. 5). After ∼60 min, 8.6% of the PLAP and 15.5% of the gp114 were internalized when compared with the total amount of bound antibodies. This relative low rate of internalization of cross-linked PLAP and gp114 has been reported before for GPI-anchored proteins and gp114 (Le Bivic et al. 1993; Deckert et al. 1996; Mayor et al. 1998). After 60 min of endocytosis and exocytosis (through recycling and/or transcytosis) the proteins have reached steady-state equilibrium (Fig. 5). LDLR-CT22 was internalized much faster and reached a maximum of ∼70% between 20 and 30 min of internalization (Fig. 5).


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)

PLAP and gp114 endocytosis from the apical membrane. Internalization of PLAP, gp114, and LDLR-CT22 from the apical plasma membrane at 37°C measured by 125I-labeled secondary antibodies (see also inset for a better view of PLAP and gp114). Numbers are expressed as percentage counts internalized compared with the total bound and internalized.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: PLAP and gp114 endocytosis from the apical membrane. Internalization of PLAP, gp114, and LDLR-CT22 from the apical plasma membrane at 37°C measured by 125I-labeled secondary antibodies (see also inset for a better view of PLAP and gp114). Numbers are expressed as percentage counts internalized compared with the total bound and internalized.
Mentions: Next we studied the internalization of cross-linked raft proteins from the apical plasma membrane. After cross-linking of the marker proteins at the apical surface and incubation at 37°C, we indeed observed that they were endocytosed. PLAP and gp114 were internalized slowly from the apical membrane (Fig. 5). After ∼60 min, 8.6% of the PLAP and 15.5% of the gp114 were internalized when compared with the total amount of bound antibodies. This relative low rate of internalization of cross-linked PLAP and gp114 has been reported before for GPI-anchored proteins and gp114 (Le Bivic et al. 1993; Deckert et al. 1996; Mayor et al. 1998). After 60 min of endocytosis and exocytosis (through recycling and/or transcytosis) the proteins have reached steady-state equilibrium (Fig. 5). LDLR-CT22 was internalized much faster and reached a maximum of ∼70% between 20 and 30 min of internalization (Fig. 5).

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