Peter S. McPherson

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

THE mechanisms through which synaptic vesicle membranes are reinternalized after exocytosis remain a matter of debate1–5. Because several vesicular transport steps require GTP hydrolysis6–9, GTP-γS may help identify intermediates in synaptic vesicle recycling. In GTP-γS-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 templates10,11. Dynamin is a GTPase implicated in synaptic vesicle endocytosis12–14 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.

The function of dynamin in endocytosis

Temperature-sensitive shibire mutants of Drosophila melanogaster become rapidly paralyzed upon a shift to the restrictive temperature, which is due to a block in synaptic vesicle endocytosis. The shibire gene encodes the GTPase dynamin. Recent studies have shown that dynamin forms rings at the neck of invaginated clathrin-coated pits, and have suggested that a conformational change in the ring, which correlates with GTP hydrolysis, plays an essential role in vesicle fission.

Recycling of Synaptic Vesicles

Publisher Summary A widely accepted model describes the synaptic vesicle cycle as a modification of the receptor-mediated recycling pathway present in all cells. This pathway, by which cell surface receptors, like transferrin or low-density lipoprotein receptors, are internalized and recycled back to the surface, involves two distinct vesicular transport steps: clathrin-mediated budding from the plasma membrane and fusion with early endosomes and budding from endosomes of vesicles destined to the plasma membrane. The membrane composition of synaptic vesicles and of clathrin-coated vesicles purified from synaptosomes is very similar. The clathrin adaptor complex AP2, a component of the plasma membrane-derived clathrin coat, binds to synaptotagmin. The paralytic shibire Drosophilu mutants , whose nerve terminals are depleted of synaptic vesicles at the restrictive temperature due to selective impairment of synaptic vesicle endocytosis, harbor mutations in dynamin, a guanosine triphosphatase (GTPase) implicated in clathrin-mediated endocytosis. Fission of clathrin-coated pits to form free vesicles requires dynamin. Dynamin was originally described as a microtubule-binding protein, but its function in synaptic vesicle recycling was revealed when the similarity to the protein encoded by the Drosophilu gene shibire was discovered. Until recently, most studies on synaptic vesicle recycling have focused on the role of proteins. There is increasing evidence, however, for a role of lipids and of phosphoinositides in particular, in synaptic vesicle endocytosis.