Inhibition of dynamin completely blocks compensatory synaptic vesicle endocytosis

Abstract

The ability of synapses to sustain signal propagation relies on rapid recycling of transmitter-containing presynaptic vesicles. Clathrinand dynamin-mediated retrieval of vesicular membrane has an undisputed role in synaptic vesicle recycling. There is also evidence for other modes of vesicle retrieval, including bulk retrieval and the so-called kiss-and-run recycling. Whether dynamin in required for these other modes of synaptic vesicle endocytosis remains unclear. Here, we have tested the role of dynamin in synaptic vesicle endocytosis by using a small molecule called dynasore, which rapidly inhibits the GTPase activity of dynamin with high specificity. Endocytosis after sustained or brief stimuli was completely and reversibly blocked by dynasore in cultured hippocampal neurons expressing the fluorescent tracer synaptopHluorin. By contrast, dynasore had no effect on exocytosis. In the presence of dynasore, low-frequency stimulation led to sustained accumulation of synaptopHluorin and other vesicular proteins on the surface membrane at a rate predicted from net exocytosis. These vesicular components remained on surface membranes even after the stimulus was terminated, suggesting that all endocytic events rely on dynamin during low-frequency activity as well as in the period after it. Ultrastructural analysis revealed a reduction in the density of synaptic vesicles and the presence of endocytic structures only at synapses that were stimulated in the presence of dynasore. In sum, our data indicate that dynamin is essential for all forms of compensatory synaptic vesicle endocytosis including any kiss-andrun events.clathrin ͉ hippocampal ͉ vesicle recycling ͉ kiss-and-run D espite substantial progress in understanding the mechanistic basis of the synaptic vesicle cycle, several fundamental questions remain. Evidence for a ''classical'' pathway of vesicle recycling through clathrin-mediated endocytosis comes from a variety of studies and techniques, and many molecular players in this process have been identified (1-4). There is also evidence for other modes of vesicle retrieval, including bulk retrieval (5, 6) and the so-called kiss-and-run recycling (7-12). Mechanistic studies of kiss-and-run recycling have been difficult, in part, because of a lack of agreement on the exact definition of this process.The final mechanical step of the separation of a budding clathrincoated vesicle from the plasma membrane relies on the GTPase dynamin (13,14). Studies using a temperature-sensitive dynamin ortholog shibire in Drosophila have indicated that dynamin function is critical for synaptic vesicle endocytosis (15, 16). If and how dynamin is involved in alternate modes of vesicle recycling such as kiss-and-run is unclear (17-22), especially at small central synapses where these modes have been proposed (8,12,23,24). At several giant synapses, the role of dynamin in synaptic vesicle endocytosis has been assayed by using nonhydrolyzable analogs of GTP such as guanosine 5Ј-[␥-thio]triphosphate (GTP␥S) or peptides that interfere wit...

Isoform and Splice-Variant Specific Functions of Dynamin-2 Revealed by Analysis of Conditional Knock-Out Cells

“…Therefore, to identify isoformand/or splice-variant-specific functions of dynamin, it would be necessary to perform isoform-specific knockout and reconstitution experiments. In support of this approach, it was recently shown (Ferguson et al, 2007) that Dyn1 KO synapses exhibited a less severe phenotype than obtained with dominant-negative dynamin mutants, such as shibire (Koenig and Ikeda, 1989) or with the dynamin inhibitor, dynasore (Newton et al, 2006). These authors also provided evidence for isoform-specific function at the synapse because reexpression of either Dyn1 or Dyn3, but only to a lesser degree by Dyn2, could rescue the defect in Dyn1 KO neurons (Ferguson et al, 2007).…”

Membrane curvature controls dynamin polymerization

“…Our findings suggest that dynamin could be recruited to the neck of closing clathrin-coated pits when their curvature is sufficiently high, and that its polymerization further constricts them to 10 nm. In vivo, dynamin appears at the clathrin-coated pits at the end of their formation (11)(12)(13), when the neck has a radius of ∼20 nm (35), close to the value of the critical radius (19 nm) we found for dynamin at 440 nM. Giant Unilamellar Vesicles.…”

Low Density Lipoprotein Receptor-related Protein 1 (LRP1) Forms a Signaling Complex with Platelet-derived Growth Factor Receptor-β in Endosomes and Regulates Activation of the MAPK Pathway

“…Recently, a potent and highly specific non-competitive inhibitor of dynamin GTPase activity was identified that blocks dynamin-dependent endocytosis in cells (30,31). We tested the ability of this inhibitor, termed dynasore, to prevent the internalization of LRP1.…”