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J. Cell Biol.,
Volume 141, Number 1, April 6, 1998 101-114
Department of Pathology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachussetts 02215
The molecular mechanisms mediating cell
surface trafficking of caveolae are unknown. Caveolae
bud from plasma membranes to form free carrier vesicles through a "pinching off" or fission process requiring
cytosol and driven by GTP hydrolysis (Schnitzer, J.E.,
P. Oh, and D.P. McIntosh. 1996. Science. 274:239-242).
Here, we use several independent techniques and functional assays ranging from cell-free to intact cell systems
to establish a function for dynamin in the formation of
transport vesicles from the endothelial cell plasma
membrane by mediating fission at the neck of caveolae. This caveolar fission requires interaction with cytosolic
dynamin as well as its hydrolysis of GTP. Expression of
dynamin in cytosol as well as purified recombinant dynamin alone supports GTP-induced caveolar fission in
a cell-free assay whereas its removal from cytosol or the
addition to the cytosol of specific antibodies for dynamin
inhibits this fission. Overexpression of mutant dynamin lacking normal GTPase activity not only inhibits GTP-induced fission and budding of caveolae but also prevents caveolae-mediated internalization of cholera toxin
B chain in intact and permeabilized endothelial cells.
Analysis of endothelium in vivo by subcellular fractionation and immunomicroscopy shows that dynamin is
concentrated on caveolae, primarily at the expected site
of action, their necks. Thus, through its ability to oligomerize, dynamin appears to form a structural collar
around the neck of caveolae that hydrolyzes GTP to
mediate internalization via the fission of caveolae from
the plasma membrane to form free transport vesicles.
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