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© The Rockefeller University Press, 0021-9525/1998//101 $5.00
The Journal of Cell Biology, Volume 141, Number 1, , 1998 101-114

Regular Articles

Dynamin at the Neck of Caveolae Mediates Their Budding to Form Transport Vesicles by GTP-driven Fission from the Plasma Membrane of Endothelium



Phil Oh, Deirdre P. McIntosh, and Jan E. Schnitzer

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.

Abbreviations used in this paper: BAEC, bovine aortic endothelial cells; BLMVEC, bovine lung microvascular endothelial cells; CT-B, cholera toxin B-chain; 5'NT, 5'-nucleotidase; P, plasma membranes; V and Vbud, purified sheared-off caveolae and GTP-budded caveolae.

Address all correspondence to Dr. Jan E. Schnitzer, BIDMC/Research North, Dept. of Pathology, 330 Brookline Ave., Boston, MA 02215. Tel.: (617) 667-3577. Fax: (617) 667-3591. E-mail: jschnitz{at}bidmc.harvard.edu

This work (except for the immunogold EM and transfection studies) was presented at the American Society for Cell Biology (ASCB) meeting in San Francisco, CA, on December 14–18, 1996, and at the ASCB meeting (including the immunogold EM and transfection studies) in Washington, DC, on December 13–17, 1997, and has appeared in abstract form (Oh, P., and J.E. Schnitzer. 1996. Mol. Biol. Cell. 7:83a; Oh, P., D.P. McIntosh, J.E. Schnitzer. 1997. Mol. Biol. Cell. 8:425a, respectively).


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