Using quantitative reconstitution to investigate multicomponent condensates

Abstract

Many biomolecular condensates are thought to form via liquid-liquid phase separation (LLPS) of multivalent macromolecules. For those that form through this mechanism, our understanding has benefitted significantly from biochemical reconstitutions of key components and activities. Reconstitutions of RNA-based condensates to date have mostly been based on relatively simple collections of molecules. However, proteomics and sequencing data indicate that natural RNA-based condensates are enriched in hundreds to thousands of different components, and genetic data suggest multiple interactions can contribute to condensate formation to varying degrees. In this perspective we describe recent progress in understanding RNA-based condensates through different levels of biochemical reconstitutions, as a means to bridge the gap between simple in vitro reconstitution and cellular analyses. Complex reconstitutions provide insight into the formation, regulation, and functions of multi-component condensates. We focus on two RNA-protein condensate case studies: stress granules and RNA processing bodies (P bodies), and examine the evidence for cooperative interactions among multiple components promoting LLPS. An important concept emerging from these studies is that composition and stoichiometry regulate biochemical activities within condensates. Based on the lessons learned from stress granules and P bodies we discuss forward-looking approaches to understand the thermodynamic relationships between condensate components, with the goal of developing predictive models of composition and material properties, and their effects on biochemical activities. We anticipate that quantitative reconstitutions will facilitate understanding of the complex thermodynamics and functions of diverse RNA-protein condensates.

Fixation Can Change the Appearance of Phase Separation in Living Cells

“…It is well-established that the dilute and concentrated phases of a LLPS system have different protein composition and concentrations (Currie and Rosen, 2022; Koga et al, 2011; Magdalena Estirado et al, 2020; Nott et al, 2015; Yewdall et al, 2021). The rate of fixation is known to vary with both factors by orders of magnitude, with the timescale of fixation ranging from seconds to hours (Hoffman et al, 2015; Kamps et al, 2019; Metz et al, 2006; Metz et al, 2004).…”

Quantitative reconstitution of yeast RNA processing bodies

“…In this study we found that reconstituting P bodies in vitro with all of the highly-concentrated proteins resulted in condensates with partitioning and dynamics values of individual components in quantitative agreement with cellular P bodies (39). Reconstitutions using single components, or more generally using arbitrarily-reduced complexity, often still form condensates in vitro , but these condensates are distinct from cellular condensates in terms of their composition and/or material properties (62). While we have not yet assayed the function of our in vitro P bodies, we suggest that this level of reconstitution – including three enzymes and many regulators of these enzymes – will allow for the examination of multiple relevant biochemical activities, and the interplay between these activities (see Discussion).…”

Evolved interactions stabilize many coexisting phases in multicomponent liquids

“…Our method can also be extended to describe more complex behavior of biomolecular condensates, including response to external cues [19,47], active regulation [48][49][50], and noise buffering [51,52]. Ultimately, our predictions could be tested using engineered condensates [53] and quantitative reconstitution [33]. Beside these concrete applications for biomolecular condensates, our method might also answer more fundamental questions about evolving systems: How can a cell exhibit robust functions while its proteins evolve [54][55][56]?…”

“…This showed that the number of coexisting phases typically depends on the overall composition of the system, but it is unclear how this phase count depends on the component count and the specific interaction matrix. Answering this question is critical, since typical biological condensates consist of many components [28,[31][32][33] and the scaffold-client picture [34], where a single scaffold component dominates the phase behavior, might not always apply. State-of-the art numerical techniques can simulate mixtures of up to 16 components [35,36], but these techniques are often too costly to truly explore the space of possible interactions.…”