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© The Rockefeller University Press, 0021-9525/2000//727 $5.00
The Journal of Cell Biology, Volume 148, Number 4, , 2000 727-740

Original Article

Induction of Caveolae in the Apical Plasma Membrane of Madin-Darby Canine Kidney Cells



Paul Verkadea,b, Thomas Hardera,b, Frank Lafonta,b, and Kai Simonsa,b

a European Molecular Biology Laboratory, Cell Biology and Biophysics Programme, D-69117 Heidelberg, Germany
b Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany
European Molecular Biology Laboratory, Cell Biology and Biophysics Programme, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.49-6221-38751249-6221-387330

simons{at}embl-heidelberg.de

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 (~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 (~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.

Key Words: rafts • epithelial cells • GPI-anchored proteins • caveolin • transcytosis

© 2000 The Rockefeller University Press

Abbreviations used in this paper: CD, methyl-β-cyclodextrin; DIG, detergent-insoluble glycolipid enriched complexes; FRET, fluorescence resonance energy transfer; GPI, glycosyl-phosphatidyl-inositol; HA, hemagglutinin; LCM, low carbonate medium; LDLR, low-density lipoprotein receptor; PLAP, placental alkaline phosphatase.


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