K48 and K11 polyubiquitination triggers formation of presynaptic sites. (A) Altering levels of endogenous polyUb chains by overexpressing wt and mutant Ub. (B and D) wtUb led to increased presynaptic clusters in isolated axons (B) and FM puncta along dendrites (MAP2, blue; D), which did not occur for UbK11R and UbK48R mutants. eGFP, control. Bars, 5 µm. (C) Number of presynaptic clusters per axonal length (percentage of eGFP). n, microscope FOVs. Five independent experiments. (E) Number of functional FM puncta per dendritic length (normalized to eGFP). n, dendrites. Two individual experiments. (F) Current model: a proteasome inhibition-derived polyUb nest for presynaptic assembly. PTV, piccolo-Bassoon transport vesicle. ***, P < 0.001; **, P < 0.01; and *, P < 0.05 compared with eGFP (Kruskal-Wallis test followed by the Dunn’s multiple comparison test). Results are presented as mean values ± SEM.