Figure 1. Reduced SV density and accumulation of intraterminal cisternae at Arf6 deficient synapses.
(A) Upper panels, representative electron micrographs of synaptic terminals from cultured hippocampal neurons (17 DIV) transduced with lentiviruses expressing an Arf6 shRNA (Arf6-KD) or an inactive mismatched version (Control). Scale bar, 200 nm. Lower panels, morphometric analysis of the density of SVs, docked SVs and cisternae in control (black) and Arf6-silenced (red) synapses. Data are means ± SEM from 3 independent preparations (n=134 and 162 synapses for control and Arf6-KD, respectively). (B) Upper panels, representative 3D synapse reconstructions from 60 nm-thick serial sections obtained from hippocampal neurons transduced as in A. Total SVs, docked SVs, presynaptic plasma membrane, postsynaptic density and cisternae are shown in light blue, yellow, green, blue and red respectively. Lower panels, morphometric analysis of number of SVs, docked SVs and cisternae in control (black) and Arf6-KD (red) synapses. Right panel shows the percentage of intraterminal cisternae that are connected (green) or not-connected (orange) with the plasma membrane for Arf6-KD synapses. Data are means ± SEM from 10 (Control) and 10 (Arf6-KD) reconstructed synapses. Statistical analysis was performed with the unpaired Student’s t-test. **p<0.005, ***p<0.001, versus control.
Figure 1—figure supplement 1. Synaptic localization of Arf6.
Figure 1—figure supplement 2. Tools for silencing and rescue Arf6 expression.
Figure 1—figure supplement 3. Rescue of the Arf6-KD ultrastructural phenotype.
