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 Parkinson's 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 moleculesboth 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..

Label-Free Techniques for Probing Biomolecular Condensates

“…164 11D,E). 167 They showed that even at dilute concentrations far from the concentrations needed for macroscopic assembly to be observed, these proteins nucleate and form nanoclusters that are kinetically trapped. They were able to explore the role of temperature, pH, molecular crowders, and biochemical modification across a wide range of masses spanning 50−1500 kDA.…”

“…(C–E) X-ray interferometric scattering microscopy (ISCAT) and mass photometry. Reprinted with permission from ref . Copyright 2023 Springer Nature.…”

“…The produced contrast value can be compared with a known protein mass standard to “weigh” the analyte, a technique known as mass photometry. This has been employed successfully for the detection of single proteins, nucleic acids, viruses, and other nanoscale objects. − Ray et al recently reported work that employed the same technique to study and quantify condensates formed by Alpha synuclein proteins during the nucleation and formation phase (Figure D,E) . They showed that even at dilute concentrations far from the concentrations needed for macroscopic assembly to be observed, these proteins nucleate and form nanoclusters that are kinetically trapped.…”

Biomimetic Structural Proteins: Modular Assembly and High Mechanical Performance

“…Liquid−liquid phase separation (LLPS) is critical in many physiological processes like the formation of membrane-less organelles and disease-related amyloid fibrils. 37 It is also a key process in the assembly of colloidal and biopolymer systems. During the spinning of natural silk, the precursors are preserved as the LLPS form, forming a condensed phase of protein materials that can further solidify during liquid-to-solid transition.…”

“…Various assembly techniques and inducing molecules have been involved in modular assembly, endowing the bulk material with novel optical, thermodynamic, and physiological features. Liquid–liquid phase separation (LLPS) is critical in many physiological processes like the formation of membrane-less organelles and disease-related amyloid fibrils . It is also a key process in the assembly of colloidal and biopolymer systems.…”