Mass photometric detection and quantification of nanoscale α-synuclein phase separation

Abstract

α-Synuclein (α-Syn) liquid-liquid phase separation (LLPS) leads to irreversible amyloid fibril formation associated with Parkinsons disease pathogenesis. Critical concentrations of α-Syn LLPS are relatively high under physiological solution conditions. Moreover, α-Syn exhibits delayed LLPS kinetics under certain conditions which deviates from the behaviour predicted by classical homogeneous nucleation theory. In the current body of work, using interferometric light scattering (iSCAT), also known as mass photometry, we experimentally probe that α-Syn can form nanoscale phase separated assemblies/clusters, containing tens to hundreds of molecules, both above and below the critical LLPS concentration down to physiologically relevant scales. The formation of these clusters is instantaneous, even under conditions where the formation of microscopically visible droplets takes several days. However, they account for a very small volume fraction below saturation concentration. The slow growth of the nanoclusters can be attributed to a kinetic barrier which can be overcome by increasing the solution temperature to just below the droplet melting point. We provide reasons for caution in quantifying dilute phase concentrations for α-Syn LLPS samples containing nanoscale droplets, which can only be separated using ultracentrifugation. In addition, we also delineate that the presence of certain surfaces facilitates α-Syn droplet nucleation under conditions of delayed kinetics but is not a mandatory prerequisite for nanocluster formation. Taken together, our findings reveal that phase separation of α-Syn occurs at a wider range of solution conditions than predicted so far and provides an important step towards understanding α-Syn LLPS within physiological scales.

Formation kinetics and physicochemical properties of mesoscopic Alpha-Synuclein assemblies modulated by sodium chloride and a distinct pulsed electric field

“…Similar phase diagram of ASN mixed with PEG8000 was also observed by Sawner's group 11 and Ray's lab. 22 ASN selfassemblies, without applying a crowding agent, were also observed after 24 hours of incubation (the first column of ESI † Fig. S2).…”

α-Synuclein liquid condensates fuel fibrillar α-synuclein growth

“…In vitro α-Syn droplets were generated in the presence of crowding agents or high salt concentration through LLPS and the protein was shown to acquire solid-like properties in a time-dependent manner (27)(28)(29)(30)(31). Besides, some data suggest that α-Syn can form nanoscale structures below the saturation concentration, playing a role in aggregation (32). These in vitro observations did not take into account complex biochemical phenomena that take place within cells such as promiscuous interactions, crowding within the cytosol, cellular compartmentalization, and stress (33)(34)(35).…”

Merging-limited coarsening governs long-term stability of nanoscale condensates

“…Surprisingly, recent experiments revealed that nanoscale condensates comprised of various protein species can remain stable in pure aqueous solutions without coarsening [14, 20-24]. In these cases, the above mentioned mechanisms do not apply, yet the conventional coarsening theories, namely BMC and Ostwald ripening, also fail to explain the suppressed coarsening dynamics [25, 26].…”

“…It is important to note that nanoscale condensates in cells have functional implications in a variety of biological contexts [7, 27], including postsynaptic density in neurotransmission [28], transcriptional condensates in gene transcription [26, 29, 30], and heterochromatin in chromatin compaction [31, 32]. Investigating the temporal evolution of nanoscale condensates is therefore particularly critical for unraveling the intriguingly slow coarsening behaviors, despite technical challenges that nanoscale condensates are typically beyond the detection limit of conventional experimental techniques, such as fluorescence microscope and UV-vis spectroscopy [7, 20].…”