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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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Triton X-100 insolubility after antibody cross-linking and internalization. Western blots from floatation gradients. Samples were taken from filter grown cells, without any treatment (− antibodies) or with antibody cross-linking and internalization for 1 h at 37°C (+ antibodies). The antibody against gp114 stains two bands on the blots; one of 114 and one of 55 kD (arrow). Percentage of Optiprep is indicated on top.
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Figure 1: Triton X-100 insolubility after antibody cross-linking and internalization. Western blots from floatation gradients. Samples were taken from filter grown cells, without any treatment (− antibodies) or with antibody cross-linking and internalization for 1 h at 37°C (+ antibodies). The antibody against gp114 stains two bands on the blots; one of 114 and one of 55 kD (arrow). Percentage of Optiprep is indicated on top.

Mentions: One of the most remarkable ultrastructural differences between the apical and basolateral plasma membranes in polarized MDCK cells is the absence of caveolae from the raft-enriched apical membrane (Vogel et al. 1998). Cross-linked raft markers have frequently been described to move into caveolae (Mayor et al. 1994; Fujimoto 1996; Wu et al. 1997). Thus, we decided to study the behavior of antibody cross-linked raft-associated proteins at the apical membrane. For this purpose, we used proteins with different raft affinities in an assay where proteins were cross-linked by antibodies and internalized. We have recently demonstrated that an antibody cross-linking technique can be used to study the association of proteins to rafts at the light microscopical level in BHK cells (Harder et al. 1998). We showed that raft proteins such as GPI-anchored PLAP and HA formed clusters that almost completely colocalized upon antibody cross-linking, while PLAP clusters and clusters formed by the non-raft protein LDLR or transferrin receptor segregated. As a first step we determined how our marker proteins behaved according to the Triton-insolubility criterion. Density floatation experiments of cold Triton X-100 solubilized control cells showed that PLAP floated to low density in Optiprep gradients (Fig. 1). When PLAP was cross-linked using antibodies with and without internalization for 1 h at 37°C, it floated in the same way as in untreated cells. We used the mutant LDL receptor LDLR-CT22 as a non-raft protein marker. The basolateral targeting signal is mutated in LDLR and is transported to the apical plasma membrane from where it can be endocytosed (Matter et al. 1992). LDLR-CT22 is Triton-soluble in control and antibody cross-linked conditions and stayed in the bottom fractions (Fig. 1). Gp114 is an integral membrane glycoprotein (Brändli et al. 1990; Le Bivic et al. 1990) that is mainly present at the apical membrane but can transcytose between the apical and basolateral membrane. In untreated cells, gp114 also behaved as a Triton-soluble protein (Fig. 1). In addition to the 114-kD protein, the antibodies against gp114 also recognized a 55-kD protein, which is a possible cleavage product of the 114-kD protein (Le Bivic et al. 1993). Upon antibody cross-linking the floatation pattern of gp114 changed. A minor fraction (5–10%) of the 114- and 55-kD protein floated to lower densities. We conclude that under normal conditions gp114 is not in DIGs. However, when the protein becomes clustered a small fraction acquired raft properties according to biochemical criteria. We have not investigated the possible cleavage of the 114-kD protein to the 55-kD protein further, but the proteins displayed identical Triton X-100 insolubility. In all further experiments, we did not discriminate between the 114- and 55-kD protein and refer to them as 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)

Triton X-100 insolubility after antibody cross-linking and internalization. Western blots from floatation gradients. Samples were taken from filter grown cells, without any treatment (− antibodies) or with antibody cross-linking and internalization for 1 h at 37°C (+ antibodies). The antibody against gp114 stains two bands on the blots; one of 114 and one of 55 kD (arrow). Percentage of Optiprep is indicated on top.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169379&req=5

Figure 1: Triton X-100 insolubility after antibody cross-linking and internalization. Western blots from floatation gradients. Samples were taken from filter grown cells, without any treatment (− antibodies) or with antibody cross-linking and internalization for 1 h at 37°C (+ antibodies). The antibody against gp114 stains two bands on the blots; one of 114 and one of 55 kD (arrow). Percentage of Optiprep is indicated on top.
Mentions: One of the most remarkable ultrastructural differences between the apical and basolateral plasma membranes in polarized MDCK cells is the absence of caveolae from the raft-enriched apical membrane (Vogel et al. 1998). Cross-linked raft markers have frequently been described to move into caveolae (Mayor et al. 1994; Fujimoto 1996; Wu et al. 1997). Thus, we decided to study the behavior of antibody cross-linked raft-associated proteins at the apical membrane. For this purpose, we used proteins with different raft affinities in an assay where proteins were cross-linked by antibodies and internalized. We have recently demonstrated that an antibody cross-linking technique can be used to study the association of proteins to rafts at the light microscopical level in BHK cells (Harder et al. 1998). We showed that raft proteins such as GPI-anchored PLAP and HA formed clusters that almost completely colocalized upon antibody cross-linking, while PLAP clusters and clusters formed by the non-raft protein LDLR or transferrin receptor segregated. As a first step we determined how our marker proteins behaved according to the Triton-insolubility criterion. Density floatation experiments of cold Triton X-100 solubilized control cells showed that PLAP floated to low density in Optiprep gradients (Fig. 1). When PLAP was cross-linked using antibodies with and without internalization for 1 h at 37°C, it floated in the same way as in untreated cells. We used the mutant LDL receptor LDLR-CT22 as a non-raft protein marker. The basolateral targeting signal is mutated in LDLR and is transported to the apical plasma membrane from where it can be endocytosed (Matter et al. 1992). LDLR-CT22 is Triton-soluble in control and antibody cross-linked conditions and stayed in the bottom fractions (Fig. 1). Gp114 is an integral membrane glycoprotein (Brändli et al. 1990; Le Bivic et al. 1990) that is mainly present at the apical membrane but can transcytose between the apical and basolateral membrane. In untreated cells, gp114 also behaved as a Triton-soluble protein (Fig. 1). In addition to the 114-kD protein, the antibodies against gp114 also recognized a 55-kD protein, which is a possible cleavage product of the 114-kD protein (Le Bivic et al. 1993). Upon antibody cross-linking the floatation pattern of gp114 changed. A minor fraction (5–10%) of the 114- and 55-kD protein floated to lower densities. We conclude that under normal conditions gp114 is not in DIGs. However, when the protein becomes clustered a small fraction acquired raft properties according to biochemical criteria. We have not investigated the possible cleavage of the 114-kD protein to the 55-kD protein further, but the proteins displayed identical Triton X-100 insolubility. In all further experiments, we did not discriminate between the 114- and 55-kD protein and refer to them as 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