Intracellular dynamics of ubiquitin-like 3 visualized using an inducible fluorescent timer expression system.

Exosomes are small extracellular vesicles (sEVs) secreted via multivesicular bodies (MVBs)/late endosomes and mediators of cell-cell communication. We previously reported a novel post-translational modification by ubiquitin-like 3 (UBL3). UBL3 is localized in MVBs and the plasma membrane and released outside as sEVs, including exosomes. Approximately 60% of proteins sorted in sEVs are affected by UBL3 and localized in various organelles, the plasma membrane, and the cytosol, suggesting that its dynamic movement in the cell before entering the MVBs. To examine the intracellular dynamics of UBL3, we constructed a sophisticated visualization system via fusing fluorescent timers that changed from blue to red form over time with UBL3 and by its expression under Tet-on regulation. Intriguingly, we found that after synthesis, UBL3 was initially distributed within the cytosol. Subsequently, UBL3 was localized to MVBs and the plasma membrane and finally showed predominant accumulation in MVBs. Furthermore, by super-resolution microscopy analysis, UBL3 was found to be associated with one of its substrates, α-tubulin, in the cytosol, and the complex was subsequently transported to MVBs. This spatiotemporal visualization system for UBL3 will form a basis for further studies to elucidate when and where UBL3 associates with its substrates/binding proteins before localization in MVBs.