Figure 9.

Presynaptic clustering on beads requires axonal E1-ubiquitin activation. (A) Requirement of protein ubiquitination for presynaptic clustering on beads. Incubation of beads for 2 h in the axonal side of microfluidic devices was performed in the presence or absence of the E1 ubiquitin activating enzyme inhibitor ziram (1, 2 and 5 μM) followed by immunostaining for tau (red) and Bassoon (green). Inhibition of axonal E1-mediated ubiquitination reduced clustering of presynaptic material on beads. Scale bar represents 5 μm. (B) Ratio of tau and Bassoon intensity between bead and corresponding adjacent site. Dashed line indicates 1. (C) Percentage of beads inducing accumulation of bassoon and tau on contacting axons in relation to adjacent off-sites. (B,C) Statistical significance by Kruskal-Wallis test followed by the Dunn’s multiple comparison test (***p < 0.001 and *p < 0.05 compared to 0 μM). A total of 525, 535, 421 and 414 beads (0, 1, 2 and 5 μM ziram, respectively) analyzed from five independent experiments.