Tubular membrane invaginations coated by dynamin rings are induced by GTP-γS in nerve terminals

Abstract

The mechanisms through which synaptic vesicle membranes are reinternalized after exocytosis remain a matter of debate. Because several vesicular transport steps require GTP hydrolysis, GTP-gamma S may help identify intermediates in synaptic vesicle recycling. In GTP-gamma S-treated nerve terminals, we observed tubular invaginations of the plasmalemma that were often, but not always, capped by a clathrin-coated bud. Strikingly, the walls of these tubules were decorated by transverse electron-dense rings that were morphologically similar to structures formed by dynamin around tubular templates. Dynamin is a GTPase implicated in synaptic vesicle endocytosis and here we show that the walls of these membranous tubules, but not their distal ends, were positive for dynamin immunoreactivity. These findings demonstrate that dynamin and clathrin act at different sites in the formation of endocytic vesicles. They strongly support a role for dynamin in the fission reaction and suggest that stabilization of the GTP-bound conformation of dynamin leads to tubule formation by progressive elongation of the vesicle stalk.

Chapter 13 Visualization of Dynamins

“…For example, dynamin spirals and dynamin-lipid tubes observed in vitro are similar to dynamin structures observed at the necks of invaginating pits in nerve synapses (Evergren et al, 2004;Koenig and Ikeda, 1989;Takei et al, 1995). Also the large Dnm1 structures seen in vitro coincide with the mitochondrial constriction sites seen in wild-type yeast (Bleazard et al, 1999;Ingerman et al, 2005).…”

Clathrin interacts specifically with amphiphysin and is displaced by dynamin1

“…This may have important implications for our understanding of the sequence of events in clathrin-coated vesicle formation. Immunogold electron microscopy of nerve terminals treated with GTPyS, which results in the accumulation of many constricted clathrin-coated invaginations, shows that dynamin predominantly forms rings at the collar or 'neck' of the invaginated vesicles [19]. Clathrin is not seen where dynamin forms these rings.…”

Endothelial Transcytotic Machinery Involves Supramolecular Protein–Lipid Complexes

“…Indeed, we found out that they contain dynamin, the fission GTPase (Takei 1995), and more significantly, caveolin, an integral membrane protein of the caveolar membrane, generally accepted as a caveolar marker…”

“…The samples precleared with a mouse IgG as mentioned in MA-TERIALS AND METHODS were immunoprecipitated with anti-myc antibody. The immunoprecipitates were analyzed by 5-20% SDS-PAGE, followed by Commassie blue staining or Western blotting with antibodies against some of the proteins known to be involved in targeting, fusion, and fission of vesicular carriers in other systems (Sollner, 1993a;Takei, 1995;Sollner, 1996). In our previous work (Predescu, 1993(Predescu, , 1994(Predescu, , 1998 we have demonstrated that PVs or caveolae are involved in transcytosis of proteins across the continuous microvascular endothelium.…”