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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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Antibody cross-linking of rGH0 induces basolateral clusters. x,y-confocal sections of the apical (A, C, and E) and basolateral (B, D, and F) plasma membrane of rGH0 expressing adenovirus-infected MDCK cells. Pairs A and B (without antibody cross-linking), C and D (after antibody cross-linking), and E and F (cholesterol depletion before antibody cross-linking) are from the same cells. Some parts of the apical plasma membrane are a bit out of focus since the cells are rounded up at the top. Bar, 10 μm.
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Figure 2: Antibody cross-linking of rGH0 induces basolateral clusters. x,y-confocal sections of the apical (A, C, and E) and basolateral (B, D, and F) plasma membrane of rGH0 expressing adenovirus-infected MDCK cells. Pairs A and B (without antibody cross-linking), C and D (after antibody cross-linking), and E and F (cholesterol depletion before antibody cross-linking) are from the same cells. Some parts of the apical plasma membrane are a bit out of focus since the cells are rounded up at the top. Bar, 10 μm.

Mentions: From the antibody cross-linking experiments in BHK cells we had seen that large clusters were formed with a size that could reach >1 μm as determined by immunofluorescence and electron microscopy (Harder et al. 1998). To study these properties in MDCK cells we performed the antibody cross-linking experiments on the apical and basolateral membranes. PLAP, gp114, and LDLR-CT22 are almost exclusively localized to the apical plasma membrane. The nonglycosylated form of the GPI-anchored rat growth hormone (rGH0) is, however, directed both apically and basolaterally and is raft associated at both membranes (Benting et al. 1999). We could therefore directly compare the distribution of this protein on the apical and basolateral plasma membrane. Confocal analysis showed that the staining for rGH0 was punctate at the apical plasma membrane without antibody cross-linking while the basolateral staining was continuous (Fig. 2A and Fig. B). After antibody cross-linking we could not detect a change in the staining pattern of the rGH0 clusters on the apical side. On the basolateral side (Fig. 2C and Fig. D) we observed cross-linked clusters like we had previously seen on the BHK plasma membrane (Harder et al. 1998). PLAP, gp114, and LDLR-CT22 analyzed on the apical plasma membrane gave a similar staining pattern as rGH0 before or after antibody cross-linking (data not shown). Since raft integrity is dependent on cholesterol we tested the effect of cholesterol depletion on the clustering process. Cyclodextrin (CD, 10 mM for 60 min) removes >50% of the total cholesterol content in MDCK cells (Scheiffele et al. 1998). Removal of cholesterol before antibody cross-linking did not change the staining of rGH0 on the apical side but on the basolateral side the clusters were not formed and a continuous staining was seen (Fig. 2E and Fig. F). This difference in the formation of raft clusters on the apical and basolateral membranes is intriguing. The apical plasma membrane is, however, covered by microvilli, and in x,y-sections these may give the impression of clusters. This phenomenon complicates the analysis. We therefore adopted the antibody cross-linking assay for electron microscopical analysis. While under control conditions PLAP, rGH0, gp114, and LDLR-CT22 were distributed randomly over the apical membrane, antibody cross-linking induced the formation of small (gold) clusters (Fig. 3 A, see also Fig. 4). We measured the size of the clusters for PLAP (n = 187 clusters) and LDLR-CT22 (n = 213 clusters) and plotted them in histograms (Fig. 3 B). From these histograms we found a distribution in the size range from 30 to ∼100 nm. The clusters were already formed at 4–8°C and did not increase in size after transfer to 37°C (data not shown). To ascertain that the size of the clusters was not due to the method of cross-linking, we performed control experiments. Instead of a two-step cross-linking procedure (primary and secondary antibody), a one-step (fixation and blocking after the primary antibody, n = 117 clusters) or a three-step (primary antibody, biotin-coupled secondary antibody, and a gold-coupled extravidin, n = 135 clusters) procedure was used. Amazingly, in all three cases, clusters with a maximum size of ∼100 nm were formed (Fig. 3 B). The size of the clusters, therefore, seems independent of the cross-linking procedure. Cross-linking of rGH0 and gp114 also induced the formation of these small clusters (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)

Antibody cross-linking of rGH0 induces basolateral clusters. x,y-confocal sections of the apical (A, C, and E) and basolateral (B, D, and F) plasma membrane of rGH0 expressing adenovirus-infected MDCK cells. Pairs A and B (without antibody cross-linking), C and D (after antibody cross-linking), and E and F (cholesterol depletion before antibody cross-linking) are from the same cells. Some parts of the apical plasma membrane are a bit out of focus since the cells are rounded up at the top. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169379&req=5

Figure 2: Antibody cross-linking of rGH0 induces basolateral clusters. x,y-confocal sections of the apical (A, C, and E) and basolateral (B, D, and F) plasma membrane of rGH0 expressing adenovirus-infected MDCK cells. Pairs A and B (without antibody cross-linking), C and D (after antibody cross-linking), and E and F (cholesterol depletion before antibody cross-linking) are from the same cells. Some parts of the apical plasma membrane are a bit out of focus since the cells are rounded up at the top. Bar, 10 μm.
Mentions: From the antibody cross-linking experiments in BHK cells we had seen that large clusters were formed with a size that could reach >1 μm as determined by immunofluorescence and electron microscopy (Harder et al. 1998). To study these properties in MDCK cells we performed the antibody cross-linking experiments on the apical and basolateral membranes. PLAP, gp114, and LDLR-CT22 are almost exclusively localized to the apical plasma membrane. The nonglycosylated form of the GPI-anchored rat growth hormone (rGH0) is, however, directed both apically and basolaterally and is raft associated at both membranes (Benting et al. 1999). We could therefore directly compare the distribution of this protein on the apical and basolateral plasma membrane. Confocal analysis showed that the staining for rGH0 was punctate at the apical plasma membrane without antibody cross-linking while the basolateral staining was continuous (Fig. 2A and Fig. B). After antibody cross-linking we could not detect a change in the staining pattern of the rGH0 clusters on the apical side. On the basolateral side (Fig. 2C and Fig. D) we observed cross-linked clusters like we had previously seen on the BHK plasma membrane (Harder et al. 1998). PLAP, gp114, and LDLR-CT22 analyzed on the apical plasma membrane gave a similar staining pattern as rGH0 before or after antibody cross-linking (data not shown). Since raft integrity is dependent on cholesterol we tested the effect of cholesterol depletion on the clustering process. Cyclodextrin (CD, 10 mM for 60 min) removes >50% of the total cholesterol content in MDCK cells (Scheiffele et al. 1998). Removal of cholesterol before antibody cross-linking did not change the staining of rGH0 on the apical side but on the basolateral side the clusters were not formed and a continuous staining was seen (Fig. 2E and Fig. F). This difference in the formation of raft clusters on the apical and basolateral membranes is intriguing. The apical plasma membrane is, however, covered by microvilli, and in x,y-sections these may give the impression of clusters. This phenomenon complicates the analysis. We therefore adopted the antibody cross-linking assay for electron microscopical analysis. While under control conditions PLAP, rGH0, gp114, and LDLR-CT22 were distributed randomly over the apical membrane, antibody cross-linking induced the formation of small (gold) clusters (Fig. 3 A, see also Fig. 4). We measured the size of the clusters for PLAP (n = 187 clusters) and LDLR-CT22 (n = 213 clusters) and plotted them in histograms (Fig. 3 B). From these histograms we found a distribution in the size range from 30 to ∼100 nm. The clusters were already formed at 4–8°C and did not increase in size after transfer to 37°C (data not shown). To ascertain that the size of the clusters was not due to the method of cross-linking, we performed control experiments. Instead of a two-step cross-linking procedure (primary and secondary antibody), a one-step (fixation and blocking after the primary antibody, n = 117 clusters) or a three-step (primary antibody, biotin-coupled secondary antibody, and a gold-coupled extravidin, n = 135 clusters) procedure was used. Amazingly, in all three cases, clusters with a maximum size of ∼100 nm were formed (Fig. 3 B). The size of the clusters, therefore, seems independent of the cross-linking procedure. Cross-linking of rGH0 and gp114 also induced the formation of these small clusters (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