Composition dependent phase separation underlies directional flux through the nucleolus

Abstract

Intracellular bodies such as nucleoli, Cajal bodies, and various signaling assemblies, represent membraneless organelles, or condensates, that form via liquidliquid phase separation (LLPS) 1,2 . Biomolecular interactions, particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions (IDRs), are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems 3-6 have led to the concept that a single fixed saturation concentration (C sat ) is a defining feature of endogenous LLPS 7-9 , and has been suggested as a mechanism for intracellular concentration buffering 2,7,8,10 . However, the assumption of a fixed C sat remains largely untested within living cells, where the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed C sat . As the concentration of individual components is varied, their partition coefficients change, in a manner that can be used to extract thermodynamic interaction energies, that we interpret within a framework we term polyphasic interaction thermodynamic analysis (PITA). We find that heterotypic interactions between protein and RNA components stabilize a variety of archetypal intracellular condensates, including the nucleolus, Cajal bodies, stress granules, and P bodies. These findings imply that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA processing condensates such as the nucleolus, this stoichiometric self-tuning manifests in selective exclusion of fully-assembled RNP complexes, providing a thermodynamic basis for vectorial ribosomal RNA (rRNA) flux out of the nucleolus. The PITA methodology is conceptually straightforward and readily implemented, and it can be broadly utilized to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deep understanding of the thermodynamics of multicomponent intracellular phase behavior and its interplay with nonequilibrium activity characteristic of endogenous condensates.To determine the thermodynamics of LLPS for intracellular condensates we first focused on the liquid granular component (GC) of nucleoli within HeLa cells, in particular on the protein Nucleophosmin (NPM1), which is known to be a key driver of nucleolar phase separation 11,12 . Under typical endogenous expression levels, we estimate NPM1 concentration in the nucleoplasm to be roughly C dil~4 μM; from simple binary phase separation models (i.e. Regular solution theory) 13 , this apparent saturation concentration, C sat , is expected to be fixed even under varied NPM1 expression levels (Fig. 1C, Supplemental Text). Consistent with previous studies 11 , ...

Unblending of Transcriptional Condensates in Human Repeat Expansion Disease

“…The lower C sat of mutant HOXD13 IDRs is in turn associated with an increase in TF IDR content and reduced MED1 IDR content of heterotypic co-condensates in vitro (Figures 3A-3F), and reduced co-activator-HOXD13 association in vivo (Figures 4A and 4B). This effect is consistent with recent reports that heterotypic interactions dominate phase separation of endogenous condensates, and C sat of heterotypic condensates can be modulated by physico-chemical properties of their components (Choi et al, 2019;Riback et al, 2019). Furthermore, the condensate unblending model may help explain why the (+7A) repeat expanded Hoxd13 allele is genetically a dominant negative allele (Albrecht and Mundlos, 2005;Villavicencio-Lorini et al, 2010), why the phenotype of repeat expansions is distinct from the phenotype of HOXD13 deactivating mutations (Bruneau et al, 2001;Dollé et al, 1993), and why the length of the repeat expansion correlates with disease severity (Goodman et al, 1997).…”

Epigenetic memory as a time integral over prior history of Polycomb phase separation

“…PHC1 shows similar phase separation behaviour, while it lacks a significant IDR. This is consistent with recent work showing that while weak interactions between IDRs can drive phase-separation in certain systems (39-44), a more complicated picture is emerging for multicomponent systems in living cells(28,45).…”

Physical Principles Underlying the Complex Biology of Intracellular Phase Transitions

“…A recent computational study based on the LASSI simulation engine helps generalize the concept of saturation concentration by showing how obligate heterotypic interactions can give rise to apparent saturation concentrations that depend on the slopes of tie lines in multidimensional phase diagrams (35). The simulations, which are built on the stickers-and-spacers formalism, when combined with suitable experiments (140), should enable a rigorous mapping between the numbers of distinct components and the apparent saturation concentrations for each of the components. In fact, our generalizations of c perc (see Equation 15) for an arbitrary number of stickers, as well as the findings reported by Choi et al (35) based on the LASSI engine and by Riback et al (140) based on experiments, help set the stage for connecting generalized observations from simulations of multicomponent systems to theories wherein each protein or RNA component has its own set of distinct stickers that may or may not interact with stickers on other protein or RNA molecules.…”