, 2008). These results challenged the proposed predominantly postsynaptic role for dynamin 3 at excitatory synapses. In this study, we used a genetic model to investigate the possibility of overlapping actions of dynamin 1 and 3 in presynaptic function and by extension to also gain insight into the potential contributions of dynamin 2. We show that lack of dynamin 3 alone in mice does not produce an obvious pathological phenotype, whereas the combined absence of dynamin 1 and 3 produces defects at the organism and cellular level that demonstrate an overlapping role of these two dynamin isoforms selleck inhibitor in synaptic vesicle

endocytosis. Surprisingly, the extremely low levels of neuronal dynamin accounted for by dynamin 2 appear to be sufficient to support neuronal life and synaptic transmission. These results also raise the

possibility that dynamin-independent mechanisms may allow a basic form of synaptic vesicle recycling. We have previously shown that endogenous dynamin 3 accumulated within presynaptic terminals in dynamin 1 KO neurons, possibly reflecting a buildup of endocytic intermediates and a role of dynamin 3 in their fission (Ferguson et al., 2007 and Hayashi et al., 2008). Consistent with an overlapping function of dynamin 1 and 3 in nerve terminals, antibodies that specifically recognize either dynamin 1 or dynamin 3 (Figures S1A–S1C [available online] and Figure 1E, respectively) revealed a similar subcellular localization of these two proteins in the brain, with a diffuse localization throughout the cytoplasm Bortezomib chemical structure and an accumulation at synapses (Figure 1A). If dynamin 1 and 3 perform overlapping functions, they would be expected to share at least a set of binding partners. The most prominent dynamin-binding partners contain SH3 domains that bind short motifs within the C-terminal proline-rich domain (PRD) of dynamin (Anggono and Robinson, 2007 and Slepnev et al., 1998). To our knowledge, the conservation of such interactions among dynamin 1, 2, and 3 has not previously

Digestive enzyme been investigated in side-by-side comparisons. To address this issue, the core PRD regions of the three dynamins were used as bait in GST pull-downs from brain extracts (Figure 1B). Western blotting of the affinity-purified material demonstrated that all proteins enriched on dynamin 1 PRD beads, such as BAR and F-BAR domain-containing proteins (endophilin, amphiphysin, SNX9, and syndapin), intersectin, and Grb2, were retained on the dynamin 3 PRD, whereas greater variability was observed for the material retained on the dynamin 2 PRD beads, suggesting greater similarities in the roles of dynamin 1 and 3 relative to those of dynamin 2. To directly test the function of dynamin 3, we generated a mouse dynamin 3 conditional (floxed) KO allele (Figure S1D). Mating of the heterozygous conditional KO mice to a Cre deleter strain (Lewandoski et al., 1997) yielded heterozygous KO mice that were bred to each other.