Dynamin-mediated internalization of caveolae.
The dynamins comprise an expanding family of ubiquitously expressed 100-kD GTPases that have been implicated in severing clathrin-coated pits during receptor-mediated endocytosis. Currently, it is unclear whether the different dynamin isoforms perform redundant functions or participate in distinct endocytic processes. To define the function of dynamin II in mammalian epithelial cells, we have generated and characterized peptide-specific antibodies to domains that either are unique to this isoform or conserved within the dynamin family. When microinjected into cultured hepatocytes these affinity-purified antibodies inhibited clathrin-mediated endocytosis and induced the formation of long plasmalemmal invaginations with attached clathrin-coated pits. In addition, clusters of distinct, nonclathrin-coated, flask-shaped invaginations resembling caveolae accumulated at the plasma membrane of antibody-injected cells. In support of this, caveola-mediated endocytosis of labeled cholera toxin B was inhibited in antibody-injected hepatocytes. Using immunoisolation techniques an anti-dynamin antibody isolated caveolar membranes directly from a hepatocyte postnuclear membrane fraction. Finally, double label immunofluorescence microscopy revealed a striking colocalization between dynamin and the caveolar coat protein caveolin. Thus, functional in vivo studies as well as ultrastructural and biochemical analyses indicate that dynamin mediates both clathrin-dependent endocytosis and the internalization of caveolae in mammalian cells.
Henley
J R
JR
Department of Molecular Neuroscience, Mayo Clinic, Rochester, Minnesota 55905, USA.
Krueger
E W
EW
Oswald
B J
BJ
McNiven
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001921:Brain / Q000502:physiology* / Q000648:ultrastructure; D002458:Cell Fractionation; D002477:Cells; D002478:Cells, Cultured; D003034:Coated Pits, Cell-Membrane / Q000502:physiology* / Q000648:ultrastructure; D034281:Dynamins; D004705:Endocytosis / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D020558:GTP Phosphohydrolases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D007527:Isoenzymes / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D051381:Rats; D017207:Rats, Sprague-Dawley
D000906:Antibodies; D007527:Isoenzymes; D010446:Peptide Fragments; D020558:GTP Phosphohydrolases; D034281:Dynamins