Diverse effects of fluorescent labels on alpha-synuclein condensate formation during liquid-liquid phase separation

Liquid-liquid phase separation is an emerging field of study, dedicated to understanding the mechanism and role of biomolecule assembly into membraneless organelles. One of the main methods employed in studying protein and nucleic acid droplet formation is fluorescence microscopy. Despite functioning as an excellent tool for monitoring biomolecule condensation, a few recent reports have presented possible drawbacks of using fluorescently labeled particles. It was observed that fluorescent tags could alter the process of protein liquid-liquid phase separation and even promote their aggregation. In this study, we examined the influence of three different protein labels on alpha-synuclein phase separation in vitro and determined that the changes in droplet formation were related to both the type, as well as concentration of the fluorescently tagged alpha-synuclein. Both protein-based labels (mCherry and eGFP) induced the formation of significantly larger droplets, while fluorescein-tagged alpha-synuclein generated an abundance of small condensates or noticeably inhibited the process of LLPS. The study also revealed that alpha-synuclein with protein-based labels could self-associate at much lower concentrations than its untagged counterpart, forming either large droplets or protein aggregates. •Fluorescently labeled proteins influence the liquid-liquid phase separation of alpha-synuclein.•The effect is related to the type and concentration of the labeled protein.•eGFP- and mCherry-alpha-synuclein significantly enlarge protein droplets.