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Vol. 13, Issue 1, 238-250, January 2002
Structural Cell Biology
Unit, Department of Medical Anatomy, The Panum
Institute, DK-2200 Copenhagen N, Denmark
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-
-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 = 
) 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.
Online version of this article
contains video material in Figures 3, 5, 7, and 9.
Online version available at www.molbiolcell.org.
*
Corresponding author. E-mail address: b.v.deurs{at}mai.ku.dk.
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