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 B12: Macromolecular Phase Separation IIFocus Live
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Sponsoring Units: DBIO DPOLY GSNP DSOFT Chair: Daphne Klotsa, Univ of NC - Chapel Hill; Yaojun Zhang, Princeton University |
Monday, March 15, 2021 11:30AM - 12:06PM Live |
B12.00001: Aging of Protein Condensates Invited Speaker: Louise Jawerth Liquid-like protein condensates (LLPCs) are intracellular compartments that segregate material without the use of a membrane. The liquid-like behavior of the condensates is a defining characteristic and the viscosity, surface tension and other material properties determine how segregated species diffuse into and within condensates; they, thus, critically impact the biological function of the condensates. It has become increasingly clear that LLPCs often have time-dependent material properties, and can transition to more solid, gel-like materials. Here, we present our efforts to quantify these new materials as they age in vitro. We measure the visco-elastic material properties of two proteins (FUS and PGL-3) by means of a combination of active and passive microrheology. At early times, we find that the droplets behave much like simple liquids but gradually become more elastic. Surprisingly, the changing mechanical properties can all be scaled onto a single master curve using one characteristic time scale which grows as the sample ages. This and other features we observe bear striking resemblance to glass-like materials. We consider these protein condensates as soft glassy materials with age dependent material properties that we call Maxwell glasses. To gain insight into the molecular origins of this behavior, we present electron microscopy images of the condensates at different ages. Furthermore, we demonstrate how salt concentration tunes the characteristics of the aging process. Lastly, we speculate on possible molecular origins that might lead to the dynamic arrest we observe and how such arrest could be used for modulation of cellular biochemistry. |
Monday, March 15, 2021 12:06PM - 12:18PM Live |
B12.00002: Liquid network connectivity regulates the stability and composition of biomolecular condensates with many components Jorge Espinosa, Jerelle A Joseph, Ignacio Sanchez-Burgos, Adiran Garaizar, Daan Frenkel, Rosana Collepardo-Guevara Liquid-liquid phase separation plays an important role in the spatiotemporal organization of the numerous molecular constituents of living cells, via formation of biomolecular condensates1. Using a minimal coarse-grained model that allows us to simulate thousands of interacting multivalent proteins, we provide predictive rules governing the stability and composition of multicomponent biomolecular condensates. Biomolecules that increase the molecular connectivity of condensates are present in higher concentrations because connectivity is positively correlated with stability. Greater connectivity within highly multicomponent condensates manifests in higher critical temperatures in the phase diagrams of accessible systems involving just a few components. Hence, composition of highly multicomponent condensates can be predicted from the critical points of reduced-component mixtures. Our findings2 expand the mechanisms relating phase behavior of multicomponent intracellular mixtures to critical parameters (temperature, pH, salt concentration, etc.) of the constituent biomolecules. |
Monday, March 15, 2021 12:18PM - 12:30PM Live |
B12.00003: Sequence dependence of biomolecular condensates Benjamin Weiner, Yigal Meir, Ned S Wingreen Cells are home to a host of biomolecular condensates – phase-separated droplets that lack a membrane – which organize intracellular processes in space and time. For example, ribosomes are produced in liquid droplets called nucleoli, and there is increasing evidence that droplets condense at specific regions of DNA to regulate gene expression. In addition to nonspecific interactions, phase separation depends on specific binding motifs between constituent molecules. But few rules have been established for how these specific, heterotypic interactions drive phase separation. Using lattice-polymer simulations and mean-field theory, we show that the sequence of binding motifs strongly affects a polymer's ability to phase separate, influencing both phase boundaries and condensate properties (e.g. viscosity and polymer extension). Notably, sequence primarily acts by determining the conformational entropy of self-bonding by single polymers. This establishes a new physical paradigm for biological control of phase separation. |
Monday, March 15, 2021 12:30PM - 12:42PM Live |
B12.00004: Guiding growing droplets through the cytoskeleton Thomas J Boeddeker, Kathryn Rosowski, Eric R Dufresne Phase-separation of biomolecules in cells takes place in a complex environment crossed by multiple filaments of the cytoskeleton or chromatin. Previous studies of artificial droplets forming inside a polymer gel have identified mechanisms that lead to a finite droplet size and even induce motion of droplets through gradients in network stiffness. Here, we study the interactions of stress granules, a phase-separated protein-RNA droplet in the cytosol, with the heterogeneous networks of actin and microtubules. Introducing a radial distribution function for disordered and finite systems such as a cell, we identify different interactions between stress granule and actin and microtubules giving rise to directed motion and final arrest of the droplet in microtubule rich locations. |
Monday, March 15, 2021 12:42PM - 12:54PM Live |
B12.00005: Round Tubulin Droplet Formation in Presence of Cross-linkers Sumon Sahu, Jennifer L Ross Phase separation phenomena in proteins have emerged as an important activity in biological systems in recent years. In cell, it has been found that cytoplasmic and nuclear bodies, such as nucleoli, cajal bodies, stress granules, germ granules, centrosome are membraneless organelles formed by phase separation. These organelles can spontaneously form when certain external factor is triggered. Prior work points to unstructured and highly charged domains being responsible for liquid phase separation, but proteins with a well-formed structure should not do this. Surprisingly, we have observed that tubulin, a well-characterized enzyme that polymerizes into microtubules, can display droplet-like phase separation in the presence of a small molecule crosslinker, Sulfo-SMCC, and a microtubule-associated protein, MAP65. These droplets are driven by the presence of MAP65, which co-localizes with the droplets. We expect that the sulfo-SMCC introduces multi-valency via cystine residues in the tubulin which might help in the phase transition. Prior work showed that Sulfo-SMCC can stop microtubules from annealing by crosslinking within the tubulin dimer. Although the droplets are initially liquid, they age quickly and become viscoelastic blobs that show little to no liquidity at later time. |
Monday, March 15, 2021 12:54PM - 1:06PM Live |
B12.00006: BIAPSS - comprehensive bioinformatic analysis of liquid-liquid phase separating sequences Aleksandra Elzbieta Badaczewska-Dawid, Davit Potoyan Liquid-liquid phase separation of many proteins is critical in the biological function of membrane-less organelles. Reversible nature of biomolecular PS in cells suggests that phases of proteins and nucleic acids are like to be tightly regulated. Post-translational modifications and single-residue mutations have been shown to lead to the dissolution of biomolecular droplets or phase transition into aggregated forms. Based on the wealth of experimental data, it reasonable to expect that the PS of proteins is highly sequence-specific. |
Monday, March 15, 2021 1:06PM - 1:18PM Live |
B12.00007: Cavitation controls droplet sizes in elastic media Estefania Vidal, David Zwicker Biomolecular condensates exist in the complex, crowded environments of biological cells. To understand how the elastic properties of the environment affect droplet formation and dynamics, we analyze a particular situation in which decreasing temperatures let droplets form in a soft matrix that can fracture. We show that large droplets only form when they fracture the surrounding matrix in a cavitation event. This cavitation provides an energy barrier for droplet growth, stabilizing small droplets on the mesh size, and diminishing the stochastic effects of nucleation. Consequently, the observed cavitated droplets are monodispersed and have highly correlated positions. In particular, we predict the density of cavitated droplets, which increases with fast temperature changes, similar to recent experiments. We also show how increasing the cooling rate can lead to bimodal droplet distributions, and we speculate on the effect of different stiffnesses. In summary, our theory provides a mechanism to control droplet sizes in elastic media. |
Monday, March 15, 2021 1:18PM - 1:30PM Live |
B12.00008: Hierarchical assembly encodes functional structure within liquid condensates Kamal Bhandari, Jeremy Schmit Biomolecular condensates are emerging as a common motif for cellular organelles. Due to their liquid properties, it is commonly thought that their utility arises from their role as a compartmentalization mechanism. We have studied two binary mixtures that form liquid condensates, SPOP/DAXX and poly-SUMO/poly-SIM. Using analytic theory to model condensate densities and molecular partitioning, we find that the condensate properties can be explained if the networks stabilizing the condensed phases have dramatically different structures. Furthermore, these structures impart critical functional properties to the resulting network. In the case of SPOP/DAXX condensates the structure allows the network to switch between a fluid state and a gel with arrested dynamics. In contrast, the SUMO/SIM structure provides a sensitive mechanism to recruit client molecules and even specifically select between closely related clients. We propose a general model in which functional structure can be embedded with a liquid when the molecules assemble hierarchically via interactions that vary widely in affinity. Stronger interactions provide structural specificity needed for functionality, while weaker interactions allow the molecules to condense without the risk of kinetic arrest. |
Monday, March 15, 2021 1:30PM - 1:42PM Live |
B12.00009: Systematic investigation of a two-fluid model describing ion-induced volume phase transition in polyelectrolyte gels Matan Mussel, Owen Lewis, Peter Basser, Ferenc Horkay An important feature of many charged polymers in solution is that they undergo a large and often discontinuous volume change in response to various environmental parameters, including pH, temperature, and ionic composition. This reversible transition plays an important role in various biological processes (e.g., swelling of secretory products, formation of membraneless organelles, and changing the hydraulic resistance of Xylem in plants) as well as in soft and “smart” materials (e.g., ion-exchange resins and controlled release). Here we report a systematic investigation of a two-fluid model describing the dynamic response of the coupled components (polymer network, solvent, and charged ions). The effects of crosslink density, concentration of ionized groups on the network chains, composition of the equilibrium salt solution containing both mono- and divalent cations, and the drag coefficient between polymer and solvent on the swelling degree of these gels, ion partitioning, and electric potential field are systematically investigated and compared to experimental results in sodium polyacrylate gels. |
Monday, March 15, 2021 1:42PM - 1:54PM Live |
B12.00010: Liquid-Liquid Phase Separation of Inorganic Polyphosphate; the Complexities of a Simple Model Hannah Seppala, Priya R Banerjee Inorganic polyphosphate (polyP) is a widely conserved primordial biological anion which is present in every branch of life. However, a thorough description of its biophysical properties is lacking. This fundamental research is crucial and could illuminate not only the biological role of polyP but lead to greater understanding of the biophysical properties of DNA and RNA, of which polyP forms a major component of the background. By understanding the contribution of polyP to the properties of the nucleic acids, we can better isolate base-dependent properties. This will contribute greatly to efforts such as bio-engineering and medicine. Here, we will present data depicting the phase behavior of polyP, showing that it forms a wide variety of condensates through liquid-liquid phase separation with numerous cations, including divalent salts, polyamines, and proteins. These condensates show a stunning variety of properties, ranging from gel-like to fully liquid. Most intriguingly, the majority of these condensates are reentrant, and dissolve upon concentration fluctuations of polyP or cations. This reentrant, surprisingly complex and richly diverse phase behavior is important as a model for more complex protein-RNA granules and in vivo membraneless organelles. |
Monday, March 15, 2021 1:54PM - 2:06PM Live |
B12.00011: Specific recognition and recruitment of client molecules by a liquid phase Kamal Bhandari, Jeremy D. Schmit There are large number of cellular bodies inside the cell which are formed by the condensation of large number of molecules. A small number of these molecules, called “Scaffolds”, produce the phase separated network with liquid-like properties and these networks recruit other molecules, called “Clients”, with the total composition providing functionality. We study a synthetic system of two types of scaffolds (poly-SUMO/poly-SIM) and two types of clients (SUMO/SIM). Based on theoretical modeling, we propose that molecules are highly aligned to form zipper structures and these zippers have defects in the bonding structure that allow for the subsequent formation of a network. We employ a transfer matrix formalism to compute the grand partition function of the zipper structures and to explain the curious non-monotonic client binding behavior observed in experiments. The filament microstructure of the droplet is sensitive to the valence and binding affinity of clients, which provides independent mechanisms to tune the magnitude (via the affinity) and the location (via the valence) of client recruitment. With tuned client affinities, it is possible for the network to specifically select between closely related clients in different regimes of parameter space. |
Monday, March 15, 2021 2:06PM - 2:18PM Live |
B12.00012: Computational modeling of liquid-liquid phase separation and cargo encapsulation in self-assembling microcompartments Farzaneh Mohajerani, Evan Sayer, Christopher Neil, Koe Inlow, Michael F Hagan Liquid-liquid phase separation and proteinaceous organelles are widely employed by cells for compartmentalization. Bacterial microcompartments (BMCs) are organelles in bacteria consisting of a protein shell that assembles around a complex of enzymes and reactants. In some BMC systems, intrinsically disordered proteins have been identified as scaffolding molecules that are indispensable for assembly and cargo encapsulation. However, the specific roles of scaffolding molecules, and how assembly depends on their properties, remain unclear. We develop equilibrium theory and dynamical computational model to describe assembly of a protein shell around cargo and scaffold molecules, with cargo coalescence and encapsulation provided by scaffold-mediated interactions. Our results predict that the shell size, amount of encapsulated cargo, and assembly pathways depend sensitively on properties of the scaffold, including its length and valency of scaffold-cargo interactions. We discuss implications of these results for synthetic biology efforts to target new molecules to microcompartments interiors. More broadly, the results elucidate how cells exploit coupling between self-assembly and liquid-liquid phase separation to organize their interiors. |
Monday, March 15, 2021 2:18PM - 2:30PM Live |
B12.00013: ATPase-Dependent Enzyme Activity Modulates the Dynamics of a Model Biological Condensate Sebastian Coupe, Nikta Fakhri Biological condensates are phase-separated ribonucleoprotein bodies that occur within cells. These concentrated phases are biochemical organizing centers of the cell, used to spatiotemporally control specialized biochemical reactions. Energy-dependent enzyme activity is thought to tune dynamics within condensates, with enzymes such as helicases, unfoldases, and chaperones implicated in this function. However, little direct evidence exists for energy and enzyme mediated condensate dynamics, and mechanisms for these processes remain unclear. One candidate family of proteins for regulating condensate dynamics is the DEAD-box helicase. These proteins are often found associated with cellular condensates, and mutations or deletions often affects the morphology, localization, and dynamics of their host condensates. However, how the activities of these enzymes affect the dynamics of biomolecular condensates has not been systematically explored. Here, we use the model phase-separating DEAD-box helicase LAF-1. Particle tracking shows how reconstituted LAF-1 condensates respond to different RNAs and nucleotides. By coupling these observations to biochemical data about LAF-1, we are building an understanding of how helicase activity can modulate condensate microenvironments. |
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