Caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking.
To investigate whether caveolae are involved in constitutive endocytic trafficking, we expressed N- and C- terminally green fluorescent protein (GFP)-tagged caveolin- 1 fusion proteins in HeLa, A431, and Madin-Darby canine kidney cells. The fusion proteins were shown by immunogold labeling to be sorted correctly to caveolae. By using confocal microscopy and photobleaching techniques, it was found that although intracellular structures labeled with GFP-tagged caveolin were dynamic, GFP-labeled caveolae were very immobile. However, after incubation with methyl- beta-cyclodextrin, distinct caveolae disappeared and the mobility of GFP-tagged caveolin in the plasma membrane increased. Treatment of cells with cytochalasin D caused lateral movement and aggregation of GFP-labeled caveolae. Therefore, both cholesterol and an intact actin cytoskeleton are required for the integrity of GFP-labeled caveolae. Moreover, stimulation with okadaic acid caused increased mobility and internalization of the labeled caveolae. Although the calculated mobile fraction (for t = infinity) of intracellular, GFP-tagged caveolin- associated structures was 70-90%, GFP-labeled caveolae in unstimulated cells had a mobile fraction of <20%, a value comparable to that previously reported for E-cadherin in junctional complexes. We therefore conclude that caveolae are not involved in constitutive endocytosis but represent a highly stable plasma membrane compartment anchored by the actin cytoskeleton.
Thomsen
Peter
P
Structural Cell Biology Unit, Department of Medical Anatomy, The Panum Institute, DK-2200 Copenhagen N, Denmark.
Roepstorff
Kirstine
K
Stahlhut
Martin
M
van Deurs
Bo
B
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D051242:Caveolin 1; D022461:Caveolins / Q000032:analysis / Q000235:genetics; D003505:Cyclodextrins / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D004285:Dogs; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004847:Epithelial Cells / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D018613:Microscopy, Confocal; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D019319:Okadaic Acid / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000032:analysis; D014162:Transfection; D014407:Tumor Cells, Cultured; D047392:beta-Cyclodextrins
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D003505:Cyclodextrins; D008164:Luminescent Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D049452:Green Fluorescent Proteins; D019319:Okadaic Acid; D015638:Cytochalasin D; D003513:Cycloheximide