Erratum: Mechanical Frustration of Phase Separation in the Cell Nucleus by Chromatin [Phys. Rev. Lett. 126, 258102 (2021)].

This corrects the article DOI: 10.1103/PhysRevLett.126.258102.

Condensate interfaces can accelerate protein aggregation.

Protein aggregates, formed from the assembly of aberrant, misfolded proteins, are a hallmark of neurodegenerative diseases. Disease-associated aggregates such as mutant Huntingtin polyQ inclusions, are typically enriched in p62/SQSTM1, an oligomeric protein that binds to and sequesters aberrant proteins. p62 has been suggested to sequester proteins through formation of liquid-like biomolecular condensates, but the physical mechanisms by which p62 condensates may regulate pathological protein aggregation remain unclear. Here, we use a light-inducible biomimetic condensate system to show that p62 condensates enhance coarsening of mutant polyQ aggregates through interface-mediated sequestration, which accelerates polyQ accumulation into larger aggregates. However, the resulting large aggregates accumulate polyubiquitinated proteins, which depletes free p62, ultimately suppressing further p62 condensation. This dynamic interplay between interface-mediated coarsening of solid aggregates and downstream consequences on the phase behavior of associated regulatory proteins could contribute to the onset and progression of protein aggregation diseases.

Viscoelasticity and advective flow of RNA underlies nucleolar form and function.

The nucleolus is the largest biomolecular condensate and facilitates transcription, processing, and assembly of ribosomal RNA (rRNA). Although nucleolar function is thought to require multiphase liquid-like properties, nucleolar fluidity and its connection to the highly coordinated transport and biogenesis of ribosomal subunits are poorly understood. Here, we use quantitative imaging, mathematical modeling, and pulse-chase nucleotide labeling to examine nucleolar material properties and rRNA dynamics. The mobility of rRNA is several orders of magnitude slower than that of nucleolar proteins, with rRNA steadily moving away from the transcriptional sites in a slow (∼1 Å/s), radially directed fashion. This constrained but directional mobility, together with polymer physics-based calculations, suggests that nascent rRNA forms an entangled gel, whose constant production drives outward flow. We propose a model in which progressive maturation of nascent rRNA reduces its initial entanglement, fluidizing the nucleolar periphery to facilitate the release of assembled pre-ribosomal particles.

Engineered molecular sensors for quantifying cell surface crowding.

Cells mediate interactions with the extracellular environment through a crowded assembly of transmembrane proteins, glycoproteins and glycolipids on their plasma membrane. The extent to which surface crowding modulates the biophysical interactions of ligands, receptors, and other macromolecules is poorly understood due to the lack of methods to quantify surface crowding on native cell membranes. In this work, we demonstrate that physical crowding on reconstituted membranes and live cell surfaces attenuates the effective binding affinity of macromolecules such as IgG antibodies in a surface crowding-dependent manner. We combine experiment and simulation to design a crowding sensor based on this principle that provides a quantitative readout of cell surface crowding. Our measurements reveal that surface crowding decreases IgG antibody binding by 2 to 20 fold in live cells compared to a bare membrane surface. Our sensors show that sialic acid, a negatively charged monosaccharide, contributes disproportionately to red blood cell surface crowding via electrostatic repulsion, despite occupying only ~1% of the total cell membrane by mass. We also observe significant differences in surface crowding for different cell types and find that expression of single oncogenes can both increase and decrease crowding, suggesting that surface crowding may be an indicator of both cell type and state. Our high-throughput, single-cell measurement of cell surface crowding may be combined with functional assays to enable further biophysical dissection of the cell surfaceome.

Size distributions of intracellular condensates reflect competition between coalescence and nucleation.

Phase separation of biomolecules into condensates has emerged as a mechanism for intracellular organization and affects many intracellular processes, including reaction pathways through the clustering of enzymes and pathway intermediates. Precise and rapid spatiotemporal control of reactions by condensates requires tuning of their sizes. However, the physical processes that govern the distribution of condensate sizes remain unclear. Here we show that both native and synthetic condensates display an exponential size distribution, which is captured by Monte Carlo simulations of fast nucleation followed by coalescence. In contrast, pathological aggregates exhibit a power-law size distribution. These distinct behaviours reflect the relative importance of nucleation and coalescence kinetics. We demonstrate this by utilizing a combination of synthetic and native condensates to probe the underlying physical mechanisms determining condensate size. The appearance of exponential distributions for abrupt nucleation versus power-law distributions under continuous nucleation may reflect a general principle that determines condensate size distributions.

The mechanobiology of nuclear phase separation.

The cell nucleus can be thought of as a complex, dynamic, living material, which functions to organize and protect the genome and coordinate gene expression. These functions are achieved via intricate mechanical and biochemical interactions among its myriad components, including the nuclear lamina, nuclear bodies, and the chromatin itself. While the biophysical organization of the nuclear lamina and chromatin have been thoroughly studied, the concept that liquid-liquid phase separation and related phase transitions play a role in establishing nuclear structure has emerged only recently. Phase transitions are likely to be intimately coupled to the mechanobiology of structural elements in the nucleus, but their interplay with one another is still not understood. Here, we review recent developments on the role of phase separation and mechanics in nuclear organization and discuss the functional implications in cell physiology and disease states.

Compartmentalization of telomeres through DNA-scaffolded phase separation.

Telomeres form unique nuclear compartments that prevent degradation and fusion of chromosome ends by recruiting shelterin proteins and regulating access of DNA damage repair factors. To understand how these dynamic components protect chromosome ends, we combine in vivo biophysical interrogation and in vitro reconstitution of human shelterin. We show that shelterin components form multicomponent liquid condensates with selective biomolecular partitioning on telomeric DNA. Tethering and anomalous diffusion prevent multiple telomeres from coalescing into a single condensate in mammalian cells. However, telomeres coalesce when brought into contact via an optogenetic approach. TRF1 and TRF2 subunits of shelterin drive phase separation, and their N-terminal domains specify interactions with telomeric DNA in vitro. Telomeric condensates selectively recruit telomere-associated factors and regulate access of DNA damage repair factors. We propose that shelterin mediates phase separation of telomeric chromatin, which underlies the dynamic yet persistent nature of the end-protection mechanism.

Mechanical Frustration of Phase Separation in the Cell Nucleus by Chromatin.

Liquid-liquid phase separation is a fundamental mechanism underlying subcellular organization. Motivated by the striking observation that optogenetically generated droplets in the nucleus display suppressed coarsening dynamics, we study the impact of chromatin mechanics on droplet phase separation. We combine theory and simulation to show that cross-linked chromatin can mechanically suppress droplets' coalescence and ripening, as well as quantitatively control their number, size, and placement. Our results highlight the role of the subcellular mechanical environment on condensate regulation.

A Primed Subpopulation of Bacteria Enables Rapid Expression of the Type 3 Secretion System in Pseudomonas aeruginosa.

Type 3 secretion systems (T3SS) are complex nanomachines that span the cell envelope and play a central role in the biology of Gram-negative pathogens and symbionts. In Pseudomonas aeruginosa, T3SS expression is strongly associated with human disease severity and with mortality in murine acute pneumonia models. Uniform exposure of isogenic cells to T3SS-activating signal results in heterogeneous expression of this critical virulence trait. To understand the function of such diversity, we measured the production of the T3SS master regulator ExsA and the expression of T3SS genes using fluorescent reporters. We found that heterogeneous expression of ExsA in the absence of activating signal generates a "primed" subpopulation of cells that can rapidly induce T3SS gene expression in response to signal. T3SS expression is accompanied by a reproductive trade-off as measured by increased division time of T3SS-expressing cells. Although T3SS-primed cells are a minority of the population, they compose the majority of T3SS-expressing cells for several hours following activation. The primed state therefore allows P. aeruginosa to maximize reproductive fitness while maintaining the capacity to quickly express the T3SS. As T3SS effectors can serve as shared public goods for nonproducing cells, this division of labor benefits the population as a whole. IMPORTANCE The expression of specific virulence traits is strongly associated with Pseudomonas aeruginosa's success in establishing acute infections but is thought to carry a cost for bacteria. Producing multiprotein secretion systems or motility organelles is metabolically expensive and can target a cell for recognition by innate immune system receptors that recognize structural components of the type 3 secretion system (T3SS) or flagellum. These acute virulence factors are also negatively selected when P. aeruginosa establishes chronic infections in the lung. We demonstrate a regulatory mechanism by which only a minority subpopulation of genetically identical P. aeruginosa cells is "primed" to respond to signals that turn on T3SS expression. This phenotypic heterogeneity allows the population to maximize the benefit of rapid T3SS effector production while maintaining a rapidly growing and nonexpressing reservoir of cells that perpetuates this genotype within the population.

Competing Protein-RNA Interaction Networks Control Multiphase Intracellular Organization.

Liquid-liquid phase separation (LLPS) mediates formation of membraneless condensates such as those associated with RNA processing, but the rules that dictate their assembly, substructure, and coexistence with other liquid-like compartments remain elusive. Here, we address the biophysical mechanism of this multiphase organization using quantitative reconstitution of cytoplasmic stress granules (SGs) with attached P-bodies in human cells. Protein-interaction networks can be viewed as interconnected complexes (nodes) of RNA-binding domains (RBDs), whose integrated RNA-binding capacity determines whether LLPS occurs upon RNA influx. Surprisingly, both RBD-RNA specificity and disordered segments of key proteins are non-essential, but modulate multiphase condensation. Instead, stoichiometry-dependent competition between protein networks for connecting nodes determines SG and P-body composition and miscibility, while competitive binding of unconnected proteins disengages networks and prevents LLPS. Inspired by patchy colloid theory, we propose a general framework by which competing networks give rise to compositionally specific and tunable condensates, while relative linkage between nodes underlies multiphase organization.

Liquid Nuclear Condensates Mechanically Sense and Restructure the Genome.

Phase transitions involving biomolecular liquids are a fundamental mechanism underlying intracellular organization. In the cell nucleus, liquid-liquid phase separation of intrinsically disordered proteins (IDPs) is implicated in assembly of the nucleolus, as well as transcriptional clusters, and other nuclear bodies. However, it remains unclear whether and how physical forces associated with nucleation, growth, and wetting of liquid condensates can directly restructure chromatin. Here, we use CasDrop, a novel CRISPR-Cas9-based optogenetic technology, to show that various IDPs phase separate into liquid condensates that mechanically exclude chromatin as they grow and preferentially form in low-density, largely euchromatic regions. A minimal physical model explains how this stiffness sensitivity arises from lower mechanical energy associated with deforming softer genomic regions. Targeted genomic loci can nonetheless be mechanically pulled together through surface tension-driven coalescence. Nuclear condensates may thus function as mechano-active chromatin filters, physically pulling in targeted genomic loci while pushing out non-targeted regions of the neighboring genome. VIDEO ABSTRACT.

Detection of Regional Variation in Selection Intensity within Protein-Coding Genes Using DNA Sequence Polymorphism and Divergence.

Numerous approaches have been developed to infer natural selection based on the comparison of polymorphism within species and divergence between species. These methods are especially powerful for the detection of uniform selection operating across a gene. However, empirical analyses have demonstrated that regions of protein-coding genes exhibiting clusters of amino acid substitutions are subject to different levels of selection relative to other regions of the same gene. To quantify this heterogeneity of selection within coding sequences, we developed Model Averaged Site Selection via Poisson Random Field (MASS-PRF). MASS-PRF identifies an ensemble of intragenic clustering models for polymorphic and divergent sites. This ensemble of models is used within the Poisson Random Field framework to estimate selection intensity on a site-by-site basis. Using simulations, we demonstrate that MASS-PRF has high power to detect clusters of amino acid variants in small genic regions, can reliably estimate the probability of a variant occurring at each nucleotide site in sequence data and is robust to historical demographic trends and recombination. We applied MASS-PRF to human gene polymorphism derived from the 1,000 Genomes Project and divergence data from the common chimpanzee. On the basis of this analysis, we discovered striking regional variation in selection intensity, indicative of positive or negative selection, in well-defined domains of genes that have previously been associated with neurological processing, immunity, and reproduction. We suggest that amino acid-altering substitutions within these regions likely are or have been selectively advantageous in the human lineage, playing important roles in protein function.