The maturation of liquid-like protein condensates into amyloid fibrils has been associated with several neurodegenerative diseases. However, the molecular mechanisms underlying this liquid-to-solid transition have remained largely unclear. Here we analyse the amyloid formation mediated by condensation of the low-complexity domain of hnRNPA1, a protein involved in amyotrophic lateral sclerosis. We show that phase separation and fibrillization are connected but distinct processes that are modulated by different regions of the protein sequence. By monitoring the spatial and temporal evolution of amyloid formation we demonstrate that the formation of fibrils does not occur homogeneously inside the droplets but is promoted at the interface of the condensates. We further show that coating the interface of the droplets with surfactant molecules inhibits fibril formation. Our results reveal that the interface of biomolecular condensates of hnRNPA1 promotes fibril formation, therefore suggesting interfaces as a potential novel therapeutic target against the formation of aberrant amyloids mediated by condensation.

Chaperone regulation of individual microscopic events in Aβ42 aggregation reveals the nature of active sites on amyloid fibrils.

Potential applications of extracellular vesicles (EVs) are attracting increasing interest in the fields of medicine, cosmetics, and nutrition. However, the manufacturing of EVs is currently characterized by low yields. This limitation severely hampers progress in research at the laboratory and clinical scales, as well as the realization of successful and cost‐effective EV‐based products. Moreover, the high level of heterogeneity of EVs further complicates reproducible manufacturing on a large scale. In this review, possible directions toward the scalable production of EVs are discussed. In particular, two strategies are considered: i) the optimization of upstream unit operations and ii) the exploitation of well‐established and mature technologies already in use in other industrial bioprocesses.

Biomolecular condensates require suitable control of material properties for their function. Here we apply Differential Dynamic Microscopy (DDM) to probe the material properties of an in vitro model of processing bodies consisting of out-of-equilibrium condensates formed by the DEAD-box ATPase Dhh1 in the presence of ATP and RNA. By applying this single-droplet technique we show that condensates within the same population exhibit a distribution of material properties, which are regulated on several levels. Removal of the low-complexity domains (LCDs) of the protein decreases the fluidity of the condensates. Structured RNA leads to a larger fraction of dynamically arrested condensates with respect to unstructured polyuridylic acid (polyU). Promotion of the enzymatic ATPase activity of Dhh1 reduces aging of the condensates and the formation of arrested structures, indicating that biochemical activity and material turnover can maintain fluid-like properties over time.

The mechanisms that underlie the regulation of enzymatic reactions by biomolecular condensates and how they scale with compartment size remain poorly understood. Here we use intrinsically disordered domains as building blocks to generate programmable enzymatic condensates of NADH-oxidase (NOX) with different sizes spanning from nanometers to microns. These disordered domains, derived from three distinct RNA-binding proteins, each possessing different net charge, result in the formation of condensates characterized by a comparable high local concentration of the enzyme yet within distinct environments. We show that only condensates with the highest recruitment of substrate and cofactor exhibit an increase in enzymatic activity. Notably, we observe an enhancement in enzymatic rate across a wide range of condensate sizes, from nanometers to microns, indicating that emergent properties of condensates can arise within assemblies as small as nanometers. Furthermore, we show a larger rate enhancement in smaller condensates. Our findings demonstrate the ability of condensates to modulate enzymatic reactions by creating distinct effective solvent environments compared to the surrounding solution, with implications for the design of protein-based heterogeneous biocatalysts.