Bulletin of the American Physical Society
APS March Meeting 2023
Volume 68, Number 3
Las Vegas, Nevada (March 5-10)
Virtual (March 20-22); Time Zone: Pacific Time
Session Q25: Biomolecular Condensates II - Molecular DeterminantsFocus
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Sponsoring Units: DBIO Chair: Ofer Kimchi, Princeton University Room: Room 217/218 |
Wednesday, March 8, 2023 3:00PM - 3:36PM |
Q25.00001: Visualizing the conformations and dynamics of FG-nucleoporins in situ Invited Speaker: Miao Yu Intrinsically disordered proteins (IDPs) are highly abundant in nature and play vital roles in various biological processes. Understanding their biological functions requires extracting their exact conformations and dynamics, which remains a technical challenge, especially in cells, due to the exceptional spatiotemporal heterogeneity of IDPs. Here we develop an experimental approach using site-specific fluorescent labeling of IDPs in mammalian cells paired with highly time-resolved fluorescence microscopy. We used this tool to study the sub-resolution permeability barrier of nuclear pore complexes (NPC), which is located within a small cavity of ~50 nm in diameter and comprised of many phenylalanine-glycine-rich IDPs, known as FG-nucleoporins (FG-Nups). Single-cell measurements of the distance distribution and conformational dynamics of FG-Nup98 segments combined with coarse-grained modeling allowed us to map the uncharted molecular environment inside the nanosized transport channel. We also compared the in situ fluorescence measurements with in vitro reconstituted phase-separated FG condensates and aqueous solution FG phases. We determined that the central channel of the NPC provides—in the terminology of Flory polymer theory—a "good solvent" environment. This enables the FG domain to adopt more expanded conformations in situ and thus facilitate nuclear transport. With more than 30% of the proteome being formed from IDPs, our combination of small-molecule-labeling-enabled fluorescence microscopy and molecular simulation opens a window into resolving disorder–function relationships of IDPs in cells. |
Wednesday, March 8, 2023 3:36PM - 3:48PM |
Q25.00002: The Effect of Polymer Length in Phase Separation Kasun K Gamage, Gilberto Valdes-Garcia, Casey Smith, Karina Martirosova, Michael Feig, Lisa J Lapidus Understanding the thermodynamics that drives liquid-liquid phase separation (LLPS) is quite important given the many numbers of diverse biomolecular systems undergoing this phenomenon. Regardless of the diversity, the processes underlying the formation of condensates exhibit physical similarities. Many studies have focused on condensates of long polymers, but very few systems of short polymer condensates have been observed and yet studied. Here we study a short polymer system of poly-Adenine RNA (polyAn) and peptide ([RGRGG]m) to understand the underlying thermodynamics of LLPS. We carried out MD simulations using the recently developed COCOMO coarse-grained (CG) model which revealed the possibility of condensates for lengths as short as 5-10 residues, which was then confirmed by experiment, making this one of the smallest LLPS systems yet observed. Condensation depends on polymer length and concentration, and phase boundaries were identified. A free energy model was also developed. Results show that the length dependency of condensates is driven solely by entropy of mixing and identifies a negative free energy (-ΔG) of phase separation, indicating the stability of the condensates. The simplicity of this system will provide the basis for understanding, more biologically realistic systems. |
Wednesday, March 8, 2023 3:48PM - 4:00PM |
Q25.00003: Early stages of FUS protein aggregation Abhinaw Kumar, Devarajan Thirumalai The formation of protein droplets through liquid-liquid phase separation is a fundamental process that leads to the formation of membrane organelles such as stress granules and nucleoli. Droplet formation happens through the attachment of single chains sequentially. Fused-in-Sarcoma (FUS) protein has been used as a model to study the aggregation process. The formation of FUS protein droplets is linked to various neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). A better understanding of the mechanism underlying FUS droplet formation may provide insight into different biologically essential processes. In this work, we perform computer simulation to elucidate the mechanism of the early stage of FUS protein aggregation using a coarse-grained model of intrinsically disordered protein previously developed by our group. We find that the conformation inside the droplet is heterogeneous and dynamic. The density of the FUS liquid droplet formed in our simulation matches the experimental density. |
Wednesday, March 8, 2023 4:00PM - 4:12PM |
Q25.00004: Molecular assembly lines regulate the size of active droplets Tyler S Harmon Large protein complexes are assembled from protein subunits to form a specific structure. In our previous work, we used theory to propose that assembly into the correct structure could be reliably achieved through an assembly line with a specific sequence of assembly steps. We illustrated that the assembly line can be self-organized through utilizing existing membraneless organelles. In this way, the droplet directly regulates the formation of the assembly line. |
Wednesday, March 8, 2023 4:12PM - 4:24PM |
Q25.00005: Adhesion and phase behavior of adhesive peptides from biofilms Jing Yan, Xin Huang, Rich Olson Adhesives that function in wet environments are widely used in ships, fishing industry, etc. Also, bio-adhesives are extensively used in biomedical applications where there is a need to adhere two wet surfaces. To search for better adhesive materials that function in an aqueous environment, engineers have extensively studied adhesive proteins from mussels and barnacles, leading to major insights into biological adhesion, often involving the formation of phase separated condensates. However, mussel foot proteins (mfps) are sensitive to environmental conditions including oxygen and pH, limiting their wide application. In a serendipitous discovery while studying how Vibrio cholerae biofilms adhere to surfaces, we discovered a short peptide sequence made of 57-amino acids that is majorly responsible for Vc adhesion to various abiotic surfaces. Abundant in lysine, tyrosine, tryptophane, and threonine, the sequence shares similarity with mfps but also important differences. In this talk, I am going to describe our recent progress in understanding how this unique sequence balances the tendency to LLPS and to adhere to surfaces in order to maximize its adhesive performance. |
Wednesday, March 8, 2023 4:24PM - 4:36PM |
Q25.00006: Instability of lamellar phase in non-equilibrium liquid-liquid phase separation Amit Kumar, Dan Deviri, Samuel A Safran Phase separation coupled to chemical reactions determine the properties of condensates such as cellular biomolecular condensates, domains of microbial colonies and chemically reactive systems. Standard non-reactive phase separation leads at long times, to a condensate of system size due to the interfacial tension of smaller sized domains. In contrast, chemical activity results in changes of the long-time shape, size and number of condensates. In non-equilibrium phase separation, the slow chemical kinetics of production and degradation of the constituents (proteins, RNA molecule etc.) play an antagonistic role to fast molecular diffusion (Ostwald ripning) and lead to a non-equilibrium steady state. For first order reactions, the non-equilibrium term maps to a long-range interaction (analogous to electrostatic) in the effective free energy (Li et. al., J. Stat. Mech., 053206 (2020); Deviri et. al., PNAS, 118, 25 (2021)). We consider an initially lamellar phase-separated microstructure and show that the effective long range interaction which originates in the chemical reactions, gives rise to unstable fluctuation modes that can lead to cylindrical or spherical microstructures. In addition, the results suggest that the reactions can reduce the effective interficial tension of condensates which may be related to the experimentally observed small tensions of the nucleolus (Caragine et. al., PRL, 121, 148101 (2018)). Both, the instability and the reduced surface tension, depend on a geometric shape factor that is a function of the aspect ratio of the macroscopic system. |
Wednesday, March 8, 2023 4:36PM - 4:48PM |
Q25.00007: Molecular dynamics of a FUS droplet wetting an actin filament Ulf D Schiller, James P Andrews Liquid phase-separating proteins are an active area of investigation in soft material design and the study of biomolecular condensates. Recent studies include the FUS protein, a naturally occurring biopolymer that wets actin filaments and bundles them into networks. However, such cellular biochemistry is sensitive to energetic and entropic forces and the mechanisms of wetting are not fully understood at the nanoscale level. We preset molecular dynamics simulations of FUS droplets forming from protein suspensions and of a FUS droplet spreading on an infinite actin filament. We used a modified Martini coarse grained force-field for protein-protein interactions as described in the literature [Benayad et al. (2021), J. Chem. Theory Comput. 17, 525-537] and extended it to FUS-actin interactions. We analyze the structural and energetic phenomena of liquid phase separating protein droplets in time and space. Thermodynamic and energetic properties such as surface tension, droplet-filament contact angles, spreading parameter, and cohesive and interaction energies are reported. |
Wednesday, March 8, 2023 4:48PM - 5:00PM |
Q25.00008: A reentrant transition in RNA aggregation Ofer Kimchi, Ella M King, Andrew G Pyo, Ned S Wingreen, Michael P Brenner RNA molecules aggregate under certain conditions. The resulting condensates are implicated in human neurological disorders, and can potentially be designed towards specified bulk properties in vitro. However, the mechanism for aggregation---including how aggregation properties change with sequence and environmental conditions---remains poorly understood. I will show that a multimerization-based framework for aggregation replicates known experimental and simulation-based results, and makes concrete predictions for the aggregation of unstudied sequences. Our model reveals that the driving force for aggregation is the increased configurational entropy associated with the multiplicity of ways to form bonds in the aggregate. I will show that the simplest system of RNA with a single self-complementary sticker nonetheless exhibits rich phase behavior, including repeat parity-dependent aggregation and a sequence-dependent reentrant phase transition. I will also describe an extension of this system to the case of multiple orthogonal stickers. Our work unifies and extends published results, both explaining the behavior of CAG-repeat RNA aggregates implicated in Huntington’s disease, and enabling the rational design of programmable RNA condensates. |
Wednesday, March 8, 2023 5:00PM - 5:12PM |
Q25.00009: RNA phase transitions are entropically driven by phosphate backbones and modulated by nucleotide sequences Gable Wadsworth, Priya R Banerjee, Rohit Pappu, Xiangze Zeng, Walter Zahurancik, Venkat Gopalan The biogenesis of ribonucleoprotein granules is thought to be driven by the reversible phase transitions of mixtures of RNA and proteins. Recently, GC-rich RNAs have been observed to form protein-independent foci in cells, which was conceptualized on the basis of an enthalpic model where base pairing and stacking interactions were postulated as drivers of RNA phase separation. Directly observing these transitions using temperature-controlled microscopy, we surprisingly discover that entropy rather than enthalpy drives RNA phase separation with a variety of RNA displaying system-specific Lower Critical Solution Temperatures (LCSTs) in the presence of Mg2+. This mode of phase separation cannot be ascribed to base-pairing and base-stacking, which are disfavored as temperature increases. Rather, desolvation entropy of the phosphate backbone as well as Mg2+ ion-mediated bridging interactions are the primary drivers of RNA phase separation. Further, we show that nucleobase composition tunes the intrinsic LCST by phosphate backbone as well as leads to a secondary percolation transition in the dense phase. Overall, our results suggest a framework to understand the molecular origin of RNA phase separation, opening new pathways to understand cellular responses to temperature variation. |
Wednesday, March 8, 2023 5:12PM - 5:24PM |
Q25.00010: Stability analysis of a phase-separating droplet with active reactions reveals rich dynamics Pradeep Natarajan, Andriy Goychuk, Arup K Chakraborty, Mehran Kardar Biomolecular condensates like the nucleolus, transcriptional condensates, and nuclear speckles spatially concentrate proteins and carry out irreversible reactions such as RNA transcription and splicing. To study the different qualitative dynamics that can arise in such active droplets, we consider a minimal two-species model where a conserved protein species phase-separates according to model B dynamics, while actively producing and passively interacting with a non-conserved molecular species (RNA) that slowly decays. A linear stability analysis reveals that the system relaxes to a stable pattern for most parameter settings, which can then be characterized by a Lyapunov functional. However, sustained oscillations can arise when proteins and RNA mutually repel each other, the former diffuses much faster than the latter, and the production of RNA is sufficiently fast. Our analysis correctly predicts the qualitative long-time behavior in numerical simulations of the full PDE model. In the case of sustained oscillations, our simulations reveal a spontaneous symmetry breaking of patterns due to shape instability. Our model provides a framework to map out the different qualitative dynamics of active droplets and can enable the engineering and control of such biomolecular systems. |
Wednesday, March 8, 2023 5:24PM - 5:36PM |
Q25.00011: Spatiotemporal control of condensates via oligomerization-dependent phase separation Hongbo Zhao, Amy R Strom, Yoonji Kim, Cornelis Storm, Cliff Brangwynne, Andrej Kosmrlj Biomolecular condensates which form via liquid-liquid phase separation can be involved in cellular functions such as genome organization and expression. They are often spatially regulated by tuning multivalent interactions. By changing the oligomerization state, modern optogenetic tools, such as Corelets, can enable direct spatiotemporal control of condensates. Here, we establish a thermodynamic framework for the oligomerization-dependent phase separation and a dynamic model based on Cahn-Hilliard theory to study the partitioning of condensates due to local light activation. In agreement with optogenetic experiments, simulations show that it is possible to locally induce droplet formation, move droplets, exert forces, and even actuate the reorganization of viscoelastic chromatin loci via capillary forces. The simulations may aid the design of programmable and customizable spatiotemporal control of biomolecular condensates. |
Wednesday, March 8, 2023 5:36PM - 5:48PM |
Q25.00012: Predicting biomolecular phase separation with field-theoretic simulations Joshua Lequieu, Ritvind Suketana The phase separation of biomolecules has catalyzed a surge of computational models that seek to predict phase separation from monomer sequence alone. Many recent studies have relied on particle-based models, yet computational limitations have restricted these models to approximations such as implicit solvents, Debye-Hückel electrostatics and simplistic treatments of phase equilibrium. In this work, we present a new model for biomolecular phase separation that avoids these approximations by using field-theoretic simulations. We demonstrate that field-theoretic simulations can be constructed to be formally equivalent to particle-based simulations and that both types of simulations yield identical values for the pressure and the chemical potential. Next we compare the performance of particle vs field simulations and show that field-theoretic simulations converge several orders of magnitude more quickly, despite giving identical results. This significant speedup permits our model to efficiently perform detailed biomolecular simulations with explicit solvent, detailed electrostatics, and thermodynamically rigorous phase diagrams using the Gibbs ensemble. Our model can recapitulate recent experimental data on intrinsically-disordered proteins and can examine the effects of amino acid sequence on their phase separation. Taken together, this work demonstrates that field-theoretic simulations can unlock a detailed molecular view into the physics of biomolecular phase separation. |
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