α-Synuclein (α-Syn) is an intrinsically disordered protein associated with the pathogenesis of neurodegenerative diseases, including Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy. α-Syn aggregates into amyloid fibrils, a major component of Lewy bodies, which is a marker for neuronal degeneration [1]. The aggregation of α-Syn can be initiated through liquid-liquid phase separation (LLPS) – a biophysical process involved in the formation of membraneless organelles [2]. Imitating intracellular environment requires precise control, therefore, our goal is to apply microfluidic technology for an in-depth LLPS study of α-Syn aggregation.
For this study we constructed and purified α-Syn with four fluorescent protein tags – eGFP, mCherry, mOrange, mCerulean. Initially, fluorescence microscopy was used to observe the LLPS process of each fluorescently tagged protein with wild type α-Syn. Then, we applied microfluidic technology that enables the study of rapidly generated subnanoliter volume droplets within microchannel systems [3]. We formed droplets containing α-Syn fibrils mixed with α-Syn monomer, suspended in a buffer solution, as well as with the molecular crowder polyethylene glycol (PEG), which induces LLPS. The resulting droplets were then analyzed using fluorescence microscopy.
Our study demonstrates that applying microfluidic technology to generate droplets containing α-Syn fibrils and monomers in the presence of PEG concentrates protein aggregates at the center of the droplets, compared to mixtures without a crowding agent. After separating aggregates from the droplets and using them as a seed for aggregation, the fibrils formed under LLPS conditions showed weaker aggregation compared to control and α-Syn fibrils formed without PEG in droplets. In the further steps, we will investigate the structures of α-Syn fibrils and optimize the application of microfluidic technology.