Affinity hierarchies and amphiphilic proteins underlie the co-assembly of nucleolar and heterochromatin condensates.

Nucleoli are surrounded by Pericentromeric Heterochromatin (PCH), reflecting a close spatial association between the two largest biomolecular condensates in eukaryotic nuclei. This nuclear organizational feature is highly conserved and is disrupted in diseased states like senescence, however, the mechanisms driving PCH-nucleolar association are unclear. High-resolution live imaging during early Drosophila development revealed a highly dynamic process in which PCH and nucleolar formation is coordinated and interdependent. When nucleolus assembly was eliminated by deleting the ribosomal RNA genes (rDNA), PCH showed increased compaction and subsequent reorganization to a shell-like structure. In addition, in embryos lacking rDNA, some nucleolar proteins were redistributed into new bodies or 'neocondensates,' including enrichment in the core of the PCH shell. These observations, combined with physical modeling and simulations, suggested that nucleolar-PCH associations are mediated by a hierarchy of affinities between PCH, nucleoli, and 'amphiphilic' protein(s) that interact with both nucleolar and PCH components. This result was validated by demonstrating that the depletion of one candidate amphiphile, the nucleolar protein Pitchoune, significantly reduced PCH-nucleolar associations. Together, these results unveil a dynamic program for establishing nucleolar-PCH associations during animal development, demonstrate that nucleoli are required for normal PCH organization, and identify Pitchoune as an amphiphilic molecular link that promotes PCH-nucleolar associations. Finally, we propose that disrupting affinity hierarchies between interacting condensates can liberate molecules to form neocondensates or other aberrant structures that could contribute to cellular disease phenotypes.

Affinity hierarchies and amphiphilic proteins underlie the co-assembly of nucleolar and heterochromatin condensates.

Nucleoli are surrounded by Pericentromeric Heterochromatin (PCH), reflecting a close spatial association between the two largest biomolecular condensates in eukaryotic nuclei. This nuclear organizational feature is highly conserved and is disrupted in diseased states like senescence, however, the mechanisms driving PCH-nucleolar association are unclear. High-resolution live imaging during early Drosophila development revealed a highly dynamic process in which PCH and nucleolar formation is coordinated and interdependent. When nucleolus assembly was eliminated by deleting the ribosomal RNA genes (rDNA), PCH showed increased compaction and subsequent reorganization to a shell-like structure. In addition, in embryos lacking rDNA, some nucleolar proteins were redistributed into new bodies or 'neocondensates,' including enrichment in the core of the PCH shell. These observations, combined with physical modeling and simulations, suggested that nucleolar-PCH associations are mediated by a hierarchy of affinities between PCH, nucleoli, and 'amphiphilic' protein(s) that interact with both nucleolar and PCH components. This result was validated by demonstrating that the depletion of one candidate amphiphile, the nucleolar protein Pitchoune, significantly reduced PCH-nucleolar associations. Together, these results unveil a dynamic program for establishing nucleolar-PCH associations during animal development, demonstrate that nucleoli are required for normal PCH organization, and identify Pitchoune as an amphiphilic molecular link that promotes PCH-nucleolar associations. Finally, we propose that disrupting affinity hierarchies between interacting condensates can liberate molecules to form neocondensates or other aberrant structures that could contribute to cellular disease phenotypes.

Regulation of chromatin microphase separation by binding of protein complexes.

We show evidence of the association of RNA polymerase II (RNAP) with chromatin in a core-shell organization, reminiscent of microphase separation where the cores comprise dense chromatin and the shell, RNAP and chromatin with low density. These observations motivate our physical model for the regulation of core-shell chromatin organization. Here, we model chromatin as a multiblock copolymer, comprising active and inactive regions (blocks) that are both in poor solvent and tend to be condensed in the absence of binding proteins. However, we show that the solvent quality for the active regions of chromatin can be regulated by the binding of protein complexes (e.g., RNAP and transcription factors). Using the theory of polymer brushes, we find that such binding leads to swelling of the active chromatin regions which in turn modifies the spatial organization of the inactive regions. In addition, we use simulations to study spherical chromatin micelles, whose cores comprise inactive regions and shells comprise active regions and bound protein complexes. In spherical micelles the swelling increases the number of inactive cores and controls their size. Thus, genetic modifications affecting the binding strength of chromatin-binding protein complexes may modulate the solvent quality experienced by chromatin and regulate the physical organization of the genome.

Controlling composition of coexisting phases via molecular transitions.

Phase separation and transitions among different molecular states are ubiquitous in living cells. Such transitions can be governed by local equilibrium thermodynamics or by active processes controlled by biological fuel. It remains largely unexplored how the behavior of phase-separating systems with molecular transitions differs between thermodynamic equilibrium and cases in which the detailed balance of the molecular transition rates is broken because of the presence of fuel. Here, we present a model of a phase-separating ternary mixture in which two components can convert into each other. At thermodynamic equilibrium, we find that molecular transitions can give rise to a lower dissolution temperature and thus reentrant phase behavior. Moreover, we find a discontinuous thermodynamic phase transition in the composition of the droplet phase if both converting molecules attract themselves with similar interaction strength. Breaking the detailed balance of the molecular transition leads to quasi-discontinuous changes in droplet composition by varying the fuel amount for a larger range of intermolecular interactions. Our findings showcase that phase separation with molecular transitions provides a versatile mechanism to control properties of intracellular and synthetic condensates via discontinuous switches in droplet composition.

Local thermodynamics govern formation and dissolution of Caenorhabditis elegans P granule condensates.

Membraneless compartments, also known as condensates, provide chemically distinct environments and thus spatially organize the cell. A well-studied example of condensates is P granules in the roundworm Caenorhabditis elegans that play an important role in the development of the germline. P granules are RNA-rich protein condensates that share the key properties of liquid droplets such as a spherical shape, the ability to fuse, and fast diffusion of their molecular components. An outstanding question is to what extent phase separation at thermodynamic equilibrium is appropriate to describe the formation of condensates in an active cellular environment. To address this question, we investigate the response of P granule condensates in living cells to temperature changes. We observe that P granules dissolve upon increasing the temperature and recondense upon lowering the temperature in a reversible manner. Strikingly, this temperature response can be captured by in vivo phase diagrams that are well described by a Flory-Huggins model at thermodynamic equilibrium. This finding is surprising due to active processes in a living cell. To address the impact of such active processes on intracellular phase separation, we discuss temperature heterogeneities. We show that, for typical estimates of the density of active processes, temperature represents a well-defined variable and that mesoscopic volume elements are at local thermodynamic equilibrium. Our findings provide strong evidence that P granule assembly and disassembly are governed by phase separation based on local thermal equilibria where the nonequilibrium nature of the cytoplasm is manifested on larger scales.

Liquid Phase Separation Controlled by pH.

We present a minimal model to study the effects of pH on liquid phase separation of macromolecules. Our model describes a mixture composed of water and macromolecules that exist in three different charge states and have a tendency to phase separate. This phase separation is affected by pH via a set of chemical reactions describing protonation and deprotonation of macromolecules, as well as self-ionization of water. We consider the simple case in which interactions are captured by Flory-Huggins interaction parameters corresponding to Debye screening lengths shorter than a nanometer, which is relevant to proteins inside biological cells under physiological conditions. We identify the conjugate thermodynamic variables at chemical equilibrium and discuss the effective free energy at fixed pH. First, we study phase diagrams as a function of macromolecule concentration and temperature at the isoelectric point of the macromolecules. We find a rich variety of phase diagram topologies, including multiple critical points, triple points, and first-order transition points. Second, we change the pH relative to the isoelectric point of the macromolecules and study how phase diagrams depend on pH. We find that these phase diagrams as a function of pH strongly depend on whether oppositely charged macromolecules or neutral macromolecules have a stronger tendency to phase separate. One key finding is that we predict the existence of a reentrant behavior as a function of pH. In addition, our model predicts that the region of phase separation is typically broader at the isoelectric point. This model could account for both in vitro phase separation of proteins as a function of pH and protein phase separation in yeast cells for pH values close to the isoelectric point of many cytosolic proteins.

Polar Positioning of Phase-Separated Liquid Compartments in Cells Regulated by an mRNA Competition Mechanism.

P granules are non-membrane-bound RNA-protein compartments that are involved in germline development in C. elegans. They are liquids that condense at one end of the embryo by localized phase separation, driven by gradients of polarity proteins such as the mRNA-binding protein MEX-5. To probe how polarity proteins regulate phase separation, we combined biochemistry and theoretical modeling. We reconstitute P granule-like droplets in vitro using a single protein PGL-3. By combining in vitro reconstitution with measurements of intracellular concentrations, we show that competition between PGL-3 and MEX-5 for mRNA can regulate the formation of PGL-3 droplets. Using theory, we show that, in a MEX-5 gradient, this mRNA competition mechanism can drive a gradient of P granule assembly with similar spatial and temporal characteristics to P granule assembly in vivo. We conclude that gradients of polarity proteins can position RNP granules during development by using RNA competition to regulate local phase separation.