The dynamins comprise an expanding family of ubiquitously expressed 100kD 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.
J Cell Biol 1998 April 6; 141(1):85-99
Dynamin-mediated Internalization of Caveolae.
Henley J, Krueger EW, Oswald B, and McNiven MA.

Accumulation of distinct endocytic structures in cultured hepatocytes injected with anti-dynamin antibodies. (a-c) Electron micrographs show typical membrane invaginations found at the surface of cells that were injected with the Dyn2-specific anti-Dyn2T antibody. Numerous budding profiles (small arrows) lacking clathrin coats and resembling caveolae were found at the plasma membrane (a and b). Long "chains" of vesicles separated by constrictions (arrowheads) also were found attached to the plasma membrane (b). In addition, clusters of multiple vesicular profiles were observed deeper within the cell (c, large arrows). Similar structures were found in the anti-Pan65 antibody-injected cells but not in the control-injected cells. (d and e) Electron micrographs of anti-Dyn2T (d) and anti-Pan65 (e) antibody-injected cells that were fixed and stained with ruthenium red. Dark vesicles reveal both surface (small arrows) and deep (large ar-rows) membrane invaginations that are continuous with the plasma membrane. Bars, 0.15 mm.

Table: Surface Density of Caveolae in Antibody-injected Hepatocytes

Hepatocytes were injected with heat-inactivated antibodies as controls (HI Ab) or the anti-dynamin antibodies ( aDyn Ab; anti-Pan65, n = 27 and anti-Dyn2T, n = 7) then fixed and processed for electron microscopy using ruthenium red to stain the plasma membranes as above, d and e. The surface density of caveolae in antibody-injected hepatocytes was determined from electron micrographs of complete cell profiles (n) for each set of injections. *Values are expressed as the median density +/- SEM. Using the Wilcoxin rank sum analysis this value differs significantly from the control value (P < 0.001; two-tailed test).

Microinjected anti-dynamin antibodies inhibit the internalization of FITC- cholera toxin B in living cells.
Fluorescence micrographs of cultured hepatocytes injected with purified antibodies to kinesin (a-c) as a control, or the anti-Pan65 (d-f) and anti-Dyn2T (g-i) antibodies. Cascade blue hydrazide was included in the antibody solutions to identify injected cells (a, d, and g). Injected cells were incubated with FITC-cholera toxin B (FITC-CTB) at 4C to allow binding, then rinsed and incubated in toxin-free medium for 2 h at 37C to allow internalization. Cells were incubated an additional 30 min at 37C in the absence (b, e, and h) or presence (c, f, and i) of Texas red-transferrin (TR-Tfn) then fixed for fluorescence microscopy. Asterisks mark injected cells. (a-c) In the control anti-kinesin antibody-injected cells and in the adjacent uninjected cells bright perinuclear green fluorescence (arrows) and noticeably dark nuclei indicate that caveola-mediated endocytosis of FITC-cholera toxin B has occurred. Likewise, the punctate red fluorescence in the double-labeled cells indicates that clathrin-mediated endocytosis of Texas red-transferrin was normal. In these cells the yellow fluorescence indicates a morphological overlap of the two ligands, most likely within endosomal compartments. (d-i) Cells that were injected with the anti-dynamin antibodies have little internal fluorescence when compared with the adjacent uninjected cells, indicating that the endocytic uptake of both FITC-cholera toxin B and Texas red-transferrin was inhibited. Bar, 10 mm.

Anti-dynamin antibodies prevent the caveola-mediated endocytosis of HRP-cholera toxin B, as confirmed by electron microscopy. Electron micrographs show hepatocytes that were injected with either a heat-inactivated antibody as a control (a) or the anti-Pan65 antibody (b-d). After the injections, cells were incubated with HRP-cholera toxin B as in Fig. 1, fixed, then processed for diaminobenzidine cytochemistry and electron microscopy. (a) A control-injected cell showing the internal electron-dense peroxidase reaction product that is sequestered largely within elements of the rough endoplasmic reticulum (ER) and the nuclear envelope (NE), with little remaining at the plasma membrane (PM). (b-d) Cells injected with the native anti-Pan65 antibody have little, if any HRP-cholera toxin B labeling of the endosomes, the endoplasmic reticulum, and the nuclear envelope, with most of the peroxidase reaction product residing on the plasma membrane or in numerous caveolae (arrows) and grape-like caveolar clusters. Similar observations were made in cells injected with the anti-Dyn2T antibody (not shown). Bars: (a) 0.5 mm; (b-d) 0.2 mm.