Figure 6.

α-Synuclein dimers inhibit vesicle fission and induce membrane tubulation during clathrin-mediated SV endocytosis. (A) Model of clathrin-mediated SV endocytosis, including several molecular players. Graphics generated by Jack Cook (Woods Hole Oceanographic Institution) using Cinema 4D. (B–E) Quantitative analysis of each morphologically distinct stage of CCP and CCV formation. Refer to (A) for individual stages. This analysis revealed that CC and NC dimer inhibited vesicle fission, as indicated by a selective increase in stage 3 CCPs. Similarly, and as expected, Dynasore inhibited vesicle fission. In contrast, monomeric α-synuclein inhibited a different stage of clathrin-mediated endocytosis, during clathrin uncoating (stage 4). Data reported indicate the averages per synapse, as measured from single sections. Bars represent mean ± SEM from n = 10–26 synapses, 1–3 axons. Asterisks indicate statistical significance (p < 0.05) by ANOVA, as compared to the controls. (F) Electron micrographs showing the observed morphologies of CCPs under the various experimental conditions. (Top) CC and NC dimers induced individual CCPs with longer necks (asterisks), compared to control, Dynasore, or monomer-treated synapses. (Bottom) CC and NC dimers also induced more complex budding structures extending from branched membrane tubules. Arrowheads indicate their connections with the PM. (G) Serial images (i, ii) and a 3D reconstruction (i + ii) showing a complex tubule extending from the PM ending in multiple CCPs. Arrowheads indicate the connections with the PM. (H–K) The α-synuclein dimers significantly increased the number of single and complex buds. In addition, neck length on CCPs, but not neck width, was increased with the α-synuclein dimers. Data reported indicate the averages per synapse, as measured from single sections. Bars represent mean ± SEM from n = 6–28 synapses, 1–3 axons. Asterisks indicate statistical significance (p < 0.05) by ANOVA, as compared to the controls.