Single-molecule FRET illuminates crucial molecular events of biological phase separation

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Living cells are a complex mixture of thousands of biomolecules, which participate in diverse chemical reactions within the controlled environment of various membrane-bounded organelles like endoplasmic reticulum, Golgi complex, mitochondria, and so on. Furthermore, cells employ a range of dynamic membrane-less organelles (MLOs) formed via the process of liquid-liquid phase separation, to achieve cytoplasmic compartmentalization. These protein and nucleic acids enriched MLOs or biomolecular condensates, which include nucleolus, stress granules, and P-bodies, etc., are responsible for some crucial cellular functions and are characterized by their liquid-like, dynamic, reversible nature. Extensive studies on these condensates have identified a class of proteins termed intrinsically disordered proteins/regions (IDPs/IDRs) as the primary drivers of these cellular condensates. These proteins lack a defined three-dimensional structure and exhibit large conformational heterogeneity, enabling large-scale networks of intermolecular interactions, and engendering the formation of an extensive mesh of protein molecules. This network of biomolecules is highly dynamic, resulting from the constant making and breaking of the ephemeral intermolecular contacts, and thus can easily be modulated by various cytoplasmic cues within the cells. 

Hence, these cellular assemblies are instrumental in a plethora of physiological processes within the prokaryotic and eukaryotic cells. However, the formation and dysregulation of these condensates are also associated with several pathological aspects of the cells. Insoluble deposits and aggregates of these IDPs/IDRs within neuronal cells are implicated in a range of debilitating neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and so on. Biological phase transition is a relatively young field, and the underlying principles governing these phase transitions remain poorly understood. Hence, discerning the sequence of events and molecular mechanism modulating these pathophysiological phase transitions has rapidly gained significance in the path of understanding the molecular aspects of neurodegeneration.  

Our work focuses on the prion-like intrinsically disordered low complexity region of Fused in Sarcoma (FUS-LC), a protein implicated in the pathology of ALS and FTLD. FUS-LC is a model IDP with a low-complexity amino acid sequence, rich in serine, tyrosine, glycine, and glutamine residues, and is believed to drive the phase transition of FUS. Using a highly sensitive, state-of-the-art technique, namely Förster Resonance energy transfer (FRET), in addition to other fluorescence and vibrational tools at the single-molecule level, we study the protein chain in the monomeric and the droplet phases and capture the changes in the polypeptide chain accompanying phase separation. Our results reveal the extensive conformational heterogeneity and the co-existing subpopulations of the FUS-LC polypeptide chain in both the dispersed and condensed phases, in addition to an unwinding of the polypeptide chain upon phase separation. The introduction of a disease-associated mutation resulted in further expansion of the FUS-LC polypeptide chain in the condensed phase. We hypothesize that this unraveling of the polypeptide chain leads to an enhanced multivalency, promoting a transition from intra-molecular to intermolecular contacts, which can further facilitate an accelerated aggregation of the mutant FUS-LC. This conformational unwinding, leading to the formation of a dense protein network, can be a general phenomenon in the phase separation of other IDPs/IDRs.