Bulletin of the American Physical Society
APS March Meeting 2021
Volume 66, Number 1
Monday–Friday, March 15–19, 2021; Virtual; Time Zone: Central Daylight Time, USA
Session A12: Macromolecular Phase Separation IFocus Live
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Sponsoring Units: DBIO DPOLY GSNP DSOFT Chair: Daphne Klotsa, Univ of NC - Chapel Hill; Patrick McCall, Max Planck Institute |
Monday, March 15, 2021 8:00AM - 8:36AM Live |
A12.00001: Decoding the physical principles of biomolecular phase separation Invited Speaker: Yaojun Zhang Cells possess non-membrane bound compartments, many of which form via liquid-liquid phase separation. Unlike conventional phase separation, e.g. the demixing of oil and water, the underlying interactions that drive biomolecular phase separation typically involve strong specific binding, often among multiple components. What are the physical principles that govern phase separation in such complex systems? We combined coarse-grained molecular dynamics simulations and analytical theory to investigate how the macroscopic phase boundaries and physical properties of condensates depend on the microscopic properties of the polymers and the concentration ratio between polymer species. We discovered novel phenomena in two-component associating polymer systems – prototypes of many membraneless organelles – including suppression of phase separation at equal polymer stoichiometry and a super-Arrhenius increase of condensate viscosity with binding strength. These results provide insight into the factors that control the formation and physical properties of condensates, and suggest potential cellular strategies for condensate regulation. |
Monday, March 15, 2021 8:36AM - 8:48AM Live |
A12.00002: Reentrant transitions in protein phase-separation: segregation vs. association Dan Deviri, Samuel Safran In the past decade, a large body of research has highlighted the biological significance of liquid-liquid phase separation (LLPS) in the formation of cellular, membraneless organelles. Such organelles are usually formed by a protein (“scaffold”) that phase separates in aqueous solution due to self-attractive interactions. In biological systems, additional solutes (“clients”), that bind the scaffold proteins are localized in the scaffold rich phase to form a distinct biochemical environment with unique functions. We use Flory Huggins theory to predict the phase diagrams of scaffold-client-solvent ternary solutions. We find that the phase diagram may exhibit reentrant LLPS as the interaction of the scaffold and client increases, leading to a transition from LLPS in which the scaffold and client are segregated, to LLPS in which the scaffold and client jointly phase-separate. We suggest this change of the composition of the dense phase as a possible mechanism underlying neurodegenerative diseases that show aberrant LLPS and are associated with mutations that affect the scaffold-client interaction energy. |
Monday, March 15, 2021 8:48AM - 9:00AM Live |
A12.00003: Fusion of Biomolecular Condensates Mahdiye Ijavi, Flurin Sturzenegger, Benjamin Schuler, Eric R Dufresne A popular approach to determine the interfacial tension of liquid-like macromolecular condensates is to measure the dynamics of coalescence of two droplets. While this is conceptually straightforward and seemingly simple to implement, it can be challenging to accurately determine the physical properties, as rates of fusion can be strongly affected by contact line dynamics. To identify the limitations of this technique, we compare the dynamics of fusion for droplets in contact with a surface to droplets in the bulk of a solution. We compare the resulting values of the capillary velocity to that determined through independent measurements of the surface tension by sessile drop tensiometry and particle tracking microrheology (Ijavi arXiv 2020). |
Monday, March 15, 2021 9:00AM - 9:12AM Live |
A12.00004: Novel “dark state” phosphate cluster formation in aqueous solution Joshua Straub, Mesopotamia Nowotarski, Jiaqi Lu, Matthew Helgeson, Alexej Jerschow, Songi Han Phosphates and polyphosphates play a ubiquitous role in biology, from structural components including cell membranes and bone to energy storage via ATP, but the solution phase space leading to the formation of polyphosphates is not entirely understood. We present novel studies in which the behavior of phosphates with increasing temperature via 31P NMR exhibit anomalous relaxation, indicating the presence of a novel phosphate state. Further investigations reveal that these “dark states” that have previously eluded detection to be assemblies of phosphates forming at up to a micron in size at elevated temperatures. The formation of these assemblies appears to be entropically driven, possibly via depletion interactions, producing a liquid-like condensed phase of phosphates, and persisting with a variety of different phosphate-containing species, including adenosine phosphates. We also explore the phase space dictating the formation of these assemblies and the mechanisms mediating their clustering utilizing dynamic light scattering and 31P solution NMR techniques. These results suggest that hidden phosphate assemblies can occur under biologically relevant conditions, lending more insight into the interpretation of in vitro and in vivo phosphate containing pathways and species. |
Monday, March 15, 2021 9:12AM - 9:24AM Live |
A12.00005: Non-equilibrium regulation and organization of transcriptional condensates by RNA synthesis Krishna Shrinivas, Jonathan Henninger, Ozgur Oksuz, Halima Schede, Phillip Sharp, Richard Young, Arup K Chakraborty Precise and robust expression of genes is essential to development and cell-type specificity. Recent evidence suggests that the key transcriptional proteins phase separate to form dynamic assemblies (or condensates) at particular genomic loci. How these dynamic assemblies are regulated dynamically and spatially is largely unknown. We leverage approaches from non-equilibrium statistical physics and complex coacervates to propose a model by which RNA, the product of gene expression, regulates transcriptional condensate dynamics. We find that low amounts of RNA synthesis promote condensate formation and higher amounts, such as from high levels of gene expression, dissolve condensates - providing a dual feedback mechanism. Predictions that connect perturbations of dynamic parameters to variations in condensate size and lifetime are verified by experiments in vivo. Finally, we extend our model to explore how spatial patterns of gene expression leads to long-range information transfer between sites of RNA synthesis and transcriptional condensates and predicts various non-equilibrium morphologies that are consistent with old experimental observations. |
Monday, March 15, 2021 9:24AM - 9:36AM Live |
A12.00006: Quantitative Theory for the Diffusive Exchange Dynamics of Liquid Condensates Lars Hubatsch, Louise Jawerth, Celina Love, Jonathan Bauermann, TY Dora Tang, Stefano Bo, Anthony A Hyman, Christoph Weber To unravel the biological function of phase-separated condensates it is key to develop a quantitative understanding of the physics governing the droplet dynamics. A key property of droplets is their ability to exchange material with their environment via diffusion. To date we lack a physics-based framework for the dynamics of labeled components inside and outside of liquid droplets. |
Monday, March 15, 2021 9:36AM - 9:48AM Live |
A12.00007: Surface condensation of a pioneer transcription factor on DNA Jose Morin, Sina Wittmann, Sandeep Choubey, Adam Klosin, Stefan Golfier, Anthony A Hyman, Frank Julicher, Stephan Grill Transcription factors cluster into sub-micrometer sized condensates while initiating transcription of their target genes. How do cells achieve a liquid phase of constrained size and location that emerge at a finely tuned concentration is not known. Here we investigate the role of DNA in nucleation of condensates, using the pioneer transcription factor KLF-4. We show that KLF-4 forms liquid-like condensates on the DNA surface at physiological concentrations, below the one required for Klf4 phase separation. We demonstrate that condensation occurs via a switch-like transition from a thin adsorbed layer to a thick condensed layer on DNA that is well described as a prewetting transition on a heterogeneous substrate. This phenomena is thus a form of surface condensation mediated by and limited to the DNA surface. |
Monday, March 15, 2021 9:48AM - 10:00AM Live |
A12.00008: Quantitative phase microscopy enables precise and efficient determination of biomolecular condensate composition Patrick M. McCall, Kyoohyun Kim, Anatol W. Fritsch, Juan M. Iglesias-Artola, Louise Jawerth, Jie Wang, Martine Ruer, Andrey Poznyakovskiy, Jan Peychl, Jochen Guck, Simon Alberti, Anthony A Hyman, Jan Brugués Many cellular processes rely on condensed macromolecular phases termed biomolecular condensates. Despite recent progress in measurements and theoretical descriptions of several condensate properties, an understanding of their most basic feature, composition, remains elusive. Here we combined quantitative phase microscopy and the physics of sessile droplets to measure the shape and composition of individual model condensates. This technique requires 1000-fold less material than traditional approaches, achieves a precision of better than 2 %, and does not rely on fluorescent dyes or tags, which we show can significantly alter protein phase behavior. We find that condensed-phase protein concentrations in three model condensates span a broad range, from 80 to 500 mg/ml, pointing to a natural diversity in condensate composition specified by protein sequence. In addition to salt- and temperature-dependent binodals, we also report time-resolved measurements revealing that PGL3 condensates undergo a contraction-like process during aging. This leads to doubling of the internal protein concentration coupled to condensate shrinkage. We anticipate that this new approach will enable understanding the physical properties of biomolecular condensates and their function. |
Monday, March 15, 2021 10:00AM - 10:12AM Live |
A12.00009: Elastically limited liquid-liquid phase separation Pierre Ronceray, Sheng Mao, Andrej Kosmrlj, Mikko Haataja Many intracellular bodies have been shown to be membrane-less liquid droplets that form through liquid-liquid phase separation (LLPS), both in the cytoplasm and in the nucleoplasm. In contrast to the archetypal oil-in-water demixing, the intracellular environment puts mechanical constraints to the formation of large droplets. In the cell nucleus, in particular, the elastic response of the chromatin network has been shown to oppose LLPS. Here we theoretically consider three scenarios by which LLPS can occur in such an elastic network: (i) by cavitation of large droplets that exclude the network, (ii) by forming many mesh-size-scale microdroplets in the pores of the network, and (iii) by permeating through the network and including it in large droplets. We propose simple criteria for which scenario is preferred, introducing a phase diagram controlled by the trade-off between elastic modulus, liquid-liquid surface tension, and liquid-network wetting properties. Our theory predicts the possibility of yet-unobserved mesh-size-limited liquid droplets in the cytoplasm and nucleoplasm. |
Monday, March 15, 2021 10:12AM - 10:24AM Live |
A12.00010: Phase separation controlled by molecular transitions Giacomo Bartolucci, Omar Adame-Arana, Xueping Zhao, Christoph Weber Phase separation and transitions among molecular states are ubiquitous in living cells. Such transitions can be governed by thermodynamics or actively controlled by biological fuel. It remains largely unexplored how the behavior of phase separating systems with molecular transitions differs between thermodynamic equilibrium and cases where detailed balance of the rates is broken due to the presence of fuel. Here, we derive a minimal model of a phase-separating ternary mixture where two components can convert into each other. We find that molecular transitions can lead to a lower critical dissolution temperature below which phase-separated droplets dissolve. Moreover, we find a discontinuous thermodynamic phase transition in the composition of the dense phase if both converting molecules attract themselves with similar interaction strength. Accounting for detailed-balance broken molecular transitions releases the system from Gibbs phase rule constraint, facilitating rapid changes in droplet composition by fuel quenches for a larger range of intermolecular interactions. Our findings showcase the potential of phase separation with molecular transitions as a mechanism to control properties of intra-cellular condensates via discontinuous switches in droplet composition. |
Monday, March 15, 2021 10:24AM - 10:36AM Live |
A12.00011: Liquid condensates increase potency of amyloid fibril inhibitors Thomas Michaels, Lakshminarayanan Mahadevan, Christoph Weber In living cells, liquid condensates form in the cytoplasm and nucleoplasm via phase separation and regulate physiological processes. They also regulate aberrant aggregation of amyloid fibrils, a process linked to Alzheimer's and Parkinson's diseases. In the absence of condensates it has been shown that amyloid aggregation can be inhibited by molecular chaperones and rationally designed drugs. However it remains unknown how this drug- or chaperone-mediated inhibition of amyloid fibril aggregation is affected by phase-separated condensates. Here we study the interplay between protein aggregation, its inhibition and liquid-liquid phase separation. Our key finding is that the potency of inhibitors of amyloid formation can be strongly enhanced. We show that the corresponding mechanism relies on the colocalization of inhibitors and aggregates inside the liquid condensate. We provide experimentally testable physicochemical conditions under which the increase of inhibitor potency is most pronounced. Our work highlights the role of spatio-temporal heterogeneity in curtailing aberrant protein aggregation and suggests design principles for amyloid inhibitors accounting for partitioning of drugs into liquid condensates. |
Monday, March 15, 2021 10:36AM - 10:48AM Live |
A12.00012: Characterization of the functional relevance of intranuclear transcription factor aggregates in living fly embryos Rahul Munshi, Michal Levo, Eric F. Wieschaus, Thomas Gregor Gene expression is driven by complex interactions of transcription factor (TF) molecules with the regulatory sequences of the DNA. Recent studies have pointed towards the existence of subnuclear micro-environments, rich in TF molecules, associated with active transcription sites. However, the underlying functional implications of such TF aggregation remain to be discovered. Here, we employ live imaging and quantitative analyses in early fly embryos to visualize and characterize such TF aggregates and simultaneously measure target gene activity. We show that the micro-environment of an actively expressing gene locus is strongly associated with TF aggregation and that their respective fluctuations correlate significantly. In addition, we test the influence of specific DNA regulatory sequences on the physical characteristics of the TF aggregates. Again, we observe significant correlations between strong TF DNA binding sites, aggregate formation, and transcriptional output. Using a mathematical model we explore how these molecular TF aggregates might stabilize gene activity and thus provide precision to gene regulation. |
Monday, March 15, 2021 10:48AM - 11:00AM Live |
A12.00013: Phase-separated compartments as biochemical reactors Sudarshana Laha, Thomas Michaels, Christoph Weber Living cells use phase-separated compartments to spatially organise fuel-driven chemical reactions. Understanding how such compartments control biochemical reactions is key to elucidate the functionality of, for example stress granules for the cell. It is also crucial for the biochemical communication among synthetic cells and RNA catalysis in coacervate protocells. Not much is known about the mechanisms underlying such spatial control of chemical reactions and how much the properties of chemical reactions are altered by the compartments. Here, we derive a theoretical framework to study fuel driven chemical reactions in the presence of compartments. We study two state transitions like phosphorylation via hydrolysis of ATP and enzymatic reactions. For two state transitions, we find that the ratio of phosphorylated product can be regulated by droplets by two orders of magnitude relative to the homogenous state. In the case of enzymatic reactions, we show that the initial rate of product formation can be increased by more than ten fold. Our studies quantify the enormous potential of phase separated compartments as biochemical reactors in living cells. |
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