Rapid increase in the degradation reporter intensity on beads precedes clustering. (A) Bead contact (dashed circles) rapidly increased the intensity of Ub^G76VGFP (firelut and green) with later clustering of VGluT1mCherry (red), not observed in off-bead sites (solid circles). (B and C, left) Ub^G76VGFP (B) and VGluT1mCherry (C) intensities at on- and off-beads throughout time (normalized to time 0, dashed line). ***, P < 0.001; **, P < 0.01; and *, P < 0.05 for on- versus off-bead (two-way ANOVA). n, beads and equivalent off-sites. (B and C, right) Values at time 0 and 150 min. ***, P < 0.001 (Wilcoxon paired t test). (D and G) Dynamics of Ub^G76VGFP accumulation on beads (circles) by FRAP (D) and FLIP (G) at off-bead sites and on-bead sites without or with enhanced local intensity of Ub^G76VGFP (arrows indicate bleaching). (D) Recovery after FRAP (boxes) is not complete at beads with enhanced reporter intensity. (G) Fluorescence loss (green boxes) after FLIP (gray boxes) is almost complete in all conditions. (E and H) Mean changes in Ub^G76V-GFP fluorescence after FRAP (E) and FLIP (H). incr., increase. Control ROIs, brownish traces. (E) FRAP ROI, greenish traces. Time constant (τ) does not differ, showing unaltered speed of recovery (off-bead, τ = 4.1 ± 0.4; on-bead without Ub^G76V-GFP, τ = 5.4 ± 1.0; on-bead with Ub^G76V-GFP, τ = 6.2 ± 0.6). n = 20–32. (H) FLIP ROI, grayish traces; analysis ROI, greenish traces. n = 27–32. (F) Percent Ub^G76V-GFP recovery after FRAP caused by diffusion from adjacent regions. (I, left) Percentage of Ub^G76V-GFP–retained fraction after FLIP in adjacent regions. (I, right) Mean time constant (in seconds) shows slower rate of fluorescence loss from beads with enhanced Ub^G76V-GFP. ns, not significant. (F and I) ***, P < 0.001 (Kruskal-Wallis test followed by the Dunn’s multiple comparison test). n, events. (A–I) Three independent experiments. Results are presented as mean values ± SEM. Bars, 5 µm.