Vesicles that are the same size as caveolae have been identified within the cytosol. The molecular machinery for caveolar internalization and vesiculation has been studied extensively by using GM1
as a marker. GM1
gangliosides are enriched within purified detergent-resistant caveolar membranes (11
) and can bind and concentrate the B subunit of cholera toxin (137
). Utilizing the cholera toxin-GM1
interaction as a caveolar marker, Parton et al. (115
) convincingly showed that the toxin is internalized into the cell’s interior via a caveola-mediated process largely distinct from that of clathrin-coated pits.
In addition, it has been demonstrated that this distinct caveola-based endocytic pathway differs from clathrin-mediated endocytosis in several ways. First, it is believed that ligand internalization by caveolae is significantly slower by approximately two- to fourfold (45
). Second, the protein phosphatase inhibitor okadaic acid stimulates caveola-mediated endocytosis but inhibits the formation of clathrin-coated vesicles (115
). Third, the sterol binding agent filipin has little or no effect on clathrin-mediated endocytosis, yet inhibits the internalization of caveolae (138
). Finally, activators of PKC, which do not inhibit clathrin-mediated endocytosis, prevent the formation of caveolar invaginations (150
From the wealth of observations described above, it appeared likely that additional proteins associated with caveolae could sustain the liberation of these plasma membrane invaginations into discrete vesicles in a regulated manner. A likely candidate for this scission process is the large GTPase dynamin that has been implicated in the formation of clathrin-coated vesicles. Dynamin was first identified from mammalian brain based on its ability to bind microtubules in a nucleotide-dependent manner (144
). The GTPase activity of dynamin can be stimulated in vitro through interaction with effector molecules at its proline-rich C-terminal domain. Insights into dynamin function emerged by demonstrating substantial identity with the Drosophila shibire
gene product (19
) which, at the restrictive temperature, is deficient at an early step in endocytosis, mainly, the ability to form coated vesicles at the plasma membrane (76
). In support of these studies, transient overexpression of dominant-negative GTP binding mutants of dynamin blocked clathrin-mediated endocytosis (24
). Dynamin has been localized to clathrin-coated pits at the plasma membrane in cultured cells (25
) and to the necks of membrane invaginations and clathrin-coated pits in an isolated synaptosomal preparation (160
) most recently. For a recent review on dynamin, see reference 99
From the original observations on dynamin function, it was attractive to predict that in addition to clathrin-mediated endocytosis, this mechano-enzyme may participate in multiple endocytic processes such as caveolar internalization and fluid phase uptake. In contrast to this model, one study had demonstrated that cultured cells, expressing a mutant neuronal dynamin form, were unable to perform clathrin-mediated endocytosis while other endocytic processes were actually increased (24
). From this single criterion, the authors concluded that dynamin is specifically required for clathrin-dependent endocytosis (82
Subsequent to these observations, two distinct laboratories accumulated independent results implicating dynamin in the scission of plasma membrane-attached caveolae. Initially, Schnitzer and colleagues (137
) convincingly demonstrated that fission of caveolae from the plasma membrane in a cell-free assay was stimulated through the addition of GTP but prevented by the nonhydrolyzable analog guanosine 5′-O
-(3-thiotriphosphate). Subsequent to these observations, Henley and McNiven (67
) tested the function of dynamin in vivo by microinjecting specific inhibitory antibodies into cultured murine hepatocytes. Using a functional assay and ultrastructural analysis, they found that cells injected with antidynamin antibodies did not internalize fluorescent conjugates of transferrin and accumulated long plasmalemmal invaginations with attached clathrin-coated pits. In addition to these structures, a marked accumulation of numerous plasmalemmal specializations resembling caveolae was observed. These plasmalemmal vesicles were distinct from clathrin-coated pits in that they lacked bristle-like coats, were ~65 to 75 nm in diameter, and had a characteristic omega or flask shape.
These initial findings suggested that caveolae may accumulate at the plasma membrane through the inhibition of either GTP hydrolysis or dynamin function and provided an incentive for both laboratories to pursue a direct study on the participation of dynamin in the scission of caveolae (66
). Concomitantly, these groups conducted functional studies to determine if caveola-mediated internalization of cholera toxin B subunit could be inhibited in intact or permeabilized cells by the addition of antidynamin antibodies. As for previous studies on caveolar internalization, cholera toxin B subunit was employed as a marker. Control experiments demonstrated that toxin uptake was largely independent of clathrin-mediated endocytosis in cultured epithelial cells, since depletion of cytoplasmic potassium ions did not inhibit the accumulation of the labeled toxin within perinuclear compartments but did block the internalization of fluorophore-labeled transferrin. When permeabilized or intact cells were incubated or microinjected with irrelevant antibodies and then incubated with fluorescent or horseradish peroxidase-conjugated toxin and viewed by fluorescence or electron microscopy, it was found that the toxin was readily internalized by control cells to a perinuclear compartment with little toxin labeling detected at the plasma membrane. Instead, toxin was concentrated within cytoplasmic organelles, including endocytic vesicles and, notably, elements of the rough endoplasmic reticulum (ER) and the nuclear envelope. In contrast, cells exposed to antidynamin antibodies did not transport toxin to intracellular compartments but instead concentrated toxin within numerous complex caveolar invaginations that had accumulated at the cell surface.
To support these functional observations, both laboratories tested for colocalization between dynamin and caveolae by using morphological and biochemical methods. Pan-dynamin antibodies coupled to magnetic beads were used to immunoisolate caveolar membranes from a hepatocyte postnuclear membrane fraction of cultured cells. By immunoblot analysis, most of the caveolin detected in the starting fraction was associated with the beads, while very little could be detected in the remaining nonbound fraction. To provide morphological support for this association, double-label immunofluorescence microscopy was used to show a significant colocalization of dynamin and caveolin. By electron microscopy, double immunogold labeling of ultrathin cryosections also showed a significant colocalization of these two proteins on plasmalemmal caveolae in lung endothelial cells. Thus, functional assays in combination with fluorescence microscopy and ultrastructural and biochemical analyses from two independent laboratories provided strong evidence that dynamin II participates in the scission of caveolae from the plasma membrane in addition to a role in clathrin-mediated endocytosis (Fig. ).
FIG. 2 Dynamin localizes to caveolae in cultured epithelial cells. (A) Fluorescence micrographs representing laser scanning confocal microscopy of cultured hepatocytes that were double-labeled with a monoclonal anti-caveolin-1 antibody as a marker for caveolae (more ...)
The observations described above raise several interesting questions that need to be addressed. These include defining the specifics of the dynamin-caveola interactions. Does dynamin bind directly or indirectly to caveolin? If this is a direct binding, what regions of the two proteins interact with each other? Is this binding regulated by the cell? It is attractive to predict that okadaic acid, which, as described above, stimulates caveolar internalization, may do so through an activation of dynamin function. Finally, which dynamin forms may interact with caveolae? Do only specific dynamin proteins interact with caveolae? Three distinct dynamin genes have been identified in mammals and include dynamin I (Dyn1), which appears to be expressed exclusively in neurons (109
); dynamin II (Dyn2), found in all tissues (20
); and dynamin III (Dyn3), which is restricted to the testis, brain, lung, and muscle tissues (16
). Each dynamin gene encodes four or more alternatively spliced isoforms (122
). Thus, while some epithelial tissues appear to express only the four different Dyn2 spliced variants, other tissues such as muscle, brain, lung, or testis may express over 20 different dynamin forms. It is interesting to note that some of these tissues, such as lung and muscle, which express the most dynamin forms, are also extremely rich in caveolae. Whether this coenrichment is of biological importance remains to be tested.