Measuring Early Kinetics of Liquid-Liquid Phase Separation of the Low-Complexity Domain of hnRNPA2

Student thesis: Master's Thesis


The formation of membraneless organelles via liquid-liquid phase separation (LLPS) allows the cell to rapidly respond to environmental conditions like cellular stress. This process is mediated by diverse multivalent interactions between RNA-binding proteins like hnRNPA2 and other macromolecules. However, the misregulation of phase separation has been associated with the accumulation of pathogenic aggregates in neurodegenerative diseases. Multiple approaches to study the kinetics followed by LLPS of hnRNPA2 have been reported, establishing that the formation of nuclei is required to form liquid-like droplets that give rise to membraneless organelles. Nonetheless, the role of nuclei and the mechanisms in which they drive phase separation are not understood yet. In the current study, we analyzed the effects of crowding agents and chemically crosslinked species on the kinetics followed by hnRNPA2. Moreover, we detected and measured the very first milliseconds of phase separation using stopped-flow kinetics. Our results indicate that crowding agents exert compaction upon the protein molecules decreasing the probability of their incorporation into liquid-like droplets, an effect that increases with the size of the crowder. Moreover, the addition of small crosslinked oligomers do not affect the kinetics of LLPS, demonstrating that they are not compatible with the particular nucleation mechanism. To confirm this, we followed the pre-steady state kinetics of LLPS and observed that the transition from protein in solution to liquid-like droplets occurs in milliseconds and without a lag phase. Our results rule out the role of nuclei in driving phase separation and open up the possibility that phase separation of hnRNPA2 is nucleation-independent but proceeds by spinodal decomposition. Further experiments in early kinetics in the presence of salts, RNA, crowding agents, preformed fibrils, and crosslinked species will help understand the complete mechanism underlying the LLPS of hnRNPA2.
Date of AwardJun 2020
Original languageEnglish
Awarding Institution
  • Vrije Universiteit Brussel

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