The liquid-to-solid transition of FUS is promoted by the condensate surface

Abstract

A wide range of macromolecules can undergo phase separation, forming biomolecular condensates in living cells. These membraneless organelles are typically highly dynamic, formed reversibly, and carry out essential functions in biological systems. Crucially, however, a further liquid-to-solid transition of the condensates can lead to irreversible pathological aggregation and cellular dysfunction associated with the onset and development of neurodegenerative diseases. Despite the importance of this liquid-to-solid transition of proteins, the mechanism by which it is initiated in normally functional condensates is unknown. Here we show, by measuring the changes in structure, dynamics, and mechanics in time and space, that single-component FUS condensates do not uniformly convert to a solid gel, but rather that liquid and gel phases coexist simultaneously within the same condensate, resulting in highly inhomogeneous structures. Furthermore, our results show that this transition originates at the interface between the condensate and the dilute continuous phase, and once initiated, the gelation process propagates toward the center of the condensate. To probe such spatially inhomogeneous rheology during condensate aging, we use a combination of established micropipette aspiration experiments together with two optical techniques, spatial dynamic mapping and reflective confocal dynamic speckle microscopy. These results reveal the importance of the spatiotemporal dimension of the liquid-to-solid transition and highlight the interface of biomolecular condensates as a critical element in driving pathological protein aggregation.

Multidimensional Super-Resolution Microscopy Unveils Nanoscale Surface Aggregates in the Aging of FUS Condensates

“…However, recent theories point to the possible gradual transformation of initially homogeneous FUS LLPS condensates to a liquid-core/gel-shell architecture through aging . A new experimental study supported this notion for the nonagitated aging of FUS condensates, suggesting the gradual solidification of the condensate surface over several days through a dissolution assay . Separately, tracing individual gold nanorods in TFEB condensates has indicated reduced mobilities at the condensate surface .…”

“…For example, for single-component LLPS condensates, whereas it is common to assume that each microdroplet adopts a homogeneous phase, recent theoretical and experimental work points to potential intracondensate inhomogeneity, e.g., the gradual transformation of FUS LLPS condensates to liquid-core/gel-shell multiphase architectures through aging. 19,25 The detection and elucidation of possible nanoscale heterogeneities in the often micrometer-sized condensate droplets are impeded by the ∼300 nm resolution of diffraction-limited optical microscopy. The past decade has seen substantial advances in super-resolution microscopy (SRM), including single-molecule localization microscopy (SMLM), which, by superlocalizing the positions of millions of individual molecules over consecutive camera frames, routinely achieves ∼20 nm spatial resolution.…”

“…The RNA-binding protein FUS and related proteins serve as model systems for LLPS and biomolecular condensates. − Formed through LLPS owing to intrinsically disordered low-complexity domains, FUS condensates are further known for their gradual transition from an initially liquid state to a solid or glassy state through aging, ,,, a process accelerated by mutations linked to neurodegenerative diseases. ,,, Related aging/maturation processes have been reported in numerous LLPS systems, including other neurodegenerative-disease players such as hnRNPA1, Tau, , TDP-43, and α-synuclein . However, the mechanism of such processes, as well as the overall structural and physical properties of the system, remain unclear.…”

From Fundamental Amyloid Protein Self-Assembly to Development of Bioplastics

“…The mechanisms for primary and secondary nucleation apply to both proteins and peptides, while the aggregation pathway for short peptides is slightly different and a higher reaction rate is expected, as the β-strands are directly exposed to the environment and not hindered by higher-level secondary and tertiary structures. Recent studies have shown that a wide range of proteins and peptides can first undergo a rapid liquid–liquid phase separation to form molecule-rich condensates due to weak molecular interactions, and then undergo a liquid-to-solid transition to form self-assembled fibrillar structures as a result of strong molecular interactions. − Interestingly, the transitions are rapid for short peptides such as z-FF (carboxybenzyl-protected diphenylalanine). The whole process lasts within an hour. , For longer peptides such as TPPGFF, and whole proteins such as FUS (FUsed in Sarcoma), the condensates are formed within minutes and converted to ordered solid structures within days. , As is clear from the mechanism described here, the self-assembly process is strongly related to the interaction between the protein molecules.…”

“…Recent studies have shown that a wide range of proteins and peptides can first undergo a rapid liquid–liquid phase separation to form molecule-rich condensates due to weak molecular interactions, and then undergo a liquid-to-solid transition to form self-assembled fibrillar structures as a result of strong molecular interactions. − Interestingly, the transitions are rapid for short peptides such as z-FF (carboxybenzyl-protected diphenylalanine). The whole process lasts within an hour. , For longer peptides such as TPPGFF, and whole proteins such as FUS (FUsed in Sarcoma), the condensates are formed within minutes and converted to ordered solid structures within days. , As is clear from the mechanism described here, the self-assembly process is strongly related to the interaction between the protein molecules. This interaction can be influenced by both intrinsic properties and external constraints, as discussed in the following sections.…”