Compartmentalization is a key feature of living systems. Cells are separated from their environment by a membrane, and intracellular compartments are widely used to carry out the biochemistry upon which life relies. Biomolecular condensates are transient intracellular compartments formed when molecules within the cytoplasm undergo a condensing phase transition. The transition produces a region within the cytoplasm that’s typically denser and/or more viscous than the surrounding fluid and the transition is often referred to as liquid-liquid phase separation (LLPS). Molecules co-located within a condensate can more readily react with one another and biologists have learned that the formation and eventual dissolution of biomolecular condensates is ubiquitous across life. While much has been learned about the functions facilitated by these transient organelles, there are many open questions about how the basic physics of LLPS is impacted by the complex, heterogeneous cellular environment within which LLPS occurs. This grant funds work by Jennifer Ross and Jennifer Schwarz, professors of experimental and theoretical physics, respectively, at Syracuse University to study how two specific features of the intracellular microenvironment—the presence of an enzyme-driven ‘active bath’ that modifies the local energy landscape, and the presence of viscoelastic polymers that modify the local entropy landscape—influence the formation and dissolution of protein condensates. The phrase ‘active bath’ refers to a fluid that has been perturbed from its equilibrium thermal state by some type of activity that leads to fluid regions with local fluctuations (e.g. position fluctuations of water molecules) that exceed those associated with the fluid’s overall (equilibrium) thermal state. In this project, the relevant activity is ‘background’ enzyme reactions; chemical reactions that do not directly involve the proteins that condense during LLPS, but which may nonetheless influence LLPS. The cellular entropy feature to be explored by Ross and Schwarz is the presence of a cytoskeleton, a network of viscoelastic (i.e. both viscous and elastic) protein filaments that act to constrain the motion of molecules within a cell via crowding. The team will create cytoskeletal-like networks of varying density and stiffness by using the cytoskeletal biopolymers actin and tubulin. Experiments will vary both the overall polymer density and the actin-to-tubulin ratio. Temperature and condensing-polymer concentration are two key parameters that will be used to experimentally characterize the LLPS phase transition, and Ross and Schwarz plan to study two types of condensing proteins and—for each type of condensing protein—two types of phase transition.

This grant supports efforts by  Thomas Preston, a Professor of Chemistry and of Atmospheric & Ocean Sciences at McGill University, to study whether and how aerosol droplets accelerate chemical reactions important to the rise of life on early Earth. Professor Preston and colleagues plan to bring a new level of experimental control and chemical analysis to Origin-of-Life (OoL) droplet-chemistry studies by containing droplets in a ‘trap’ that allows individual droplets to be studied for long periods of time, and by applying two powerful spectroscopic techniques (Raman spectroscopy and mass spectrometry) to study in-droplet chemistry. In-droplet chemistry may have contributed to the rise of life on Earth by accelerating various chemical reactions. This ‘acceleration of chemistry’ is important to origins-of-life theories because as chemical reaction times increase, yields become vanishingly small, and thus not useful for understanding abiogenesis. It has been reported that confinement inside droplets can accelerate chemical reactions by a factor of up to one million, but well controlled experiments supporting such claims are rare and there is considerable uncertainty about the mechanisms responsible for any enhanced chemical reactivity. Preston and his team will use use two levitation techniques – one based on light (optical trap) and another based on an electric field (electrodynamic trap) – to study individual droplets for long periods of time (hours to days) and under  a variety of well controlled conditions, to shed light on whether and how droplet environments accelerate chemical reactivity. Factors to be controlled and studied include droplet size (and thus surface-to-volume ratio) and the impact on droplet chemistry of photoelectric and electrical field excitation. Studying droplets for relatively long periods of time will allow the team to deploy highly informative measurement techniques such as Raman spectroscopy (probes molecular bonding) and mass spectrometry (information on molecular species) and to do so in a time-resolved manner.  Two different areas of droplet chemistry will be investigated: hydrogen cyanide chemistry and phosphorylation reactions, each of which include reactions essential to origin of life studies, including amide bond formation, nucleoside formation, polysaccharide synthesis, and ion-phosphate attachment. 

Biomolecular condensates are transient organelles that are ubiquitous across life, and which are widely used -for instance- to host intracellular chemistry.  The ubiquity of transient compartmentalization hints at an evolutionarily earlier time when complex chemistry and compartmentalization coupled to evolve in tandem. This grant funds Rebecca Schulman, a Professor of Chemical and Biomolecular Engineering at Johns Hopkins University, Elisa Franco, a Professor of Mechanical and Aerospace Engineering at the University of California Los Angeles, and Deborah Fygenson, a Professor of Physics at the University of California Santa Barbara, to conduct a series of in vitro studies to improve our understanding of systems where chemical reactions are coupled to condensation dynamics. What are the elementary components of, and the fundamental principles governing, a plausibly-origin-of-life-relevant ‘dynamic soup’ whereby chemistry and compartmentalization couple to achieve biological function? Schulman, et. al. plan to explore this question by studying how chemical reactions affect condensate (or droplet) volume and the dynamics of droplet size change.  They’ll then leverage that knowledge to achieve sustained cycles of condensate creation, growth, and dissolution over a range of spatial and temporal scales. Under project phase 1, the team will measure the phase behavior of various condensing nucleic acid (NA) polymers as a function of the concentration of several ‘effector’ molecules that are designed to modify the condensate state. The team seeks to determine the steady state properties of a condensing-molecule / effector system defined by a fixed concentration of effector molecules. Doing so will help them interpret the effects of rapid changes in, and non-uniform distributions of, effectors produced or consumed by various chemical reactions. Under phase 2, the team will explore whether it’s possible to achieve stabilized micron-scale condensates by coupling chemical reactions and condensation. The strategy relies on two key ideas. First, that the chemistry of interest should interfere with the tendency of droplet molecules to aggregate since this will inhibit the growth of an existing droplet. The PIs will exploit chemistry that produces growth inhibiting effectors (RNA polymers). Second, the growth inhibiting chemistry should become more effective with increased droplet size since this amounts to size-stabilizing negative feedback. The team will leverage in-droplet chemistry to synthesize the growth inhibiting RNA polymers and they expect that these polymers will be more effective in large droplets because it takes longer on average to diffuse out of large droplets than small droplets. Under phase 3, the researchers aim to build reaction-condensate systems that exhibit sustained cycles of droplet emergence, growth, and dissolution. The team will pursue two strategies. First, they’ll use a so-called ‘transcriptional oscillator’ positioned in the condensate environment (the surrounding dilute phase) to chemically synthesize a droplet-growth-inhibiting (RNA-based) effector. Second, they’ll implement a chemical feedback system featuring a growth inhibiting effector that does not inhibit growth until it diffuses out of a droplet and chemically reacts with certain molecules in the environmental. If successful, the project will provide insight into how cells exploit couplings between chemical and condensation dynamics to implement biological function, while also establishing a toolset that allows researchers to build information-bearing entities that exhibit sustained cycles of formation, growth, and dissolution.

Abiogenesis is the process whereby life arose—or can arise—from nonliving matter. Somehow an assembly of chemicals containing information polymers bootstrapped itself up into a self-replicating system that grew in complexity over time. Rather little is known about how this chemical evolution can—and likely did—occur. This grant supports David Baum and David Beebe, Professors of Botany and of Biomedical Engineering, respectively, at the University of Wisconsin-Madison, in efforts to test the hypothesis that selection at the chemical ecosystem level—rather than at the level of individual molecules in a population—can yield ecosystems of (nucleic acid) information polymers that play complementary roles in promoting their collective propagation. Existing mathematical and computational models suggest that ecosystem-scale selection was important to abiogenesis. The term ‘selection’ refers to preferential survival owing to enhanced fitness in an environment. The term ‘ecosystem-scale selection’ refers to cases where the fitness serving as a basis for selection is a property of an ecosystem; here, a collection of chemicals. Ecosystem-scale selection naturally invokes the idea of a ‘container’ since containers offer a simple way of creating chemical systems that can compete with one another. In this project the container is an aqueous droplet; specifically, droplets containing different nucleic acid-based chemical systems that compete with one another for selection based on the droplets’ propensity to propagate (make more DNA/RNA). Some droplets will be discarded while others continue on in a series of experiments that allow the ecosystem in surviving droplets to evolve. The PIs aim to test the hypothesis that droplet-scale selection can lead to ecosystems of cooperating nucleic acid polymers that are particularly good at promoting their collective propagation. Baum and Beebe will perform experiments on various DNA/RNA ecosystems with an overall plan to create droplets containing a nucleic acid based chemical ecosystem, put the droplets through a series of incubate-select-propagate cycles, and then look for a response to selection by comparing the results of ‘selection’ and ‘control’ experiments. ‘Selection’ will be based on a fluorescent signal that indicates how much DNA/RNA has been synthesized and the bottom-lower-half droplets on the brightness scale will be discarded. Propagation will be achieved by splitting selected droplets in two and then fusing each ‘offspring’ droplet with a ‘food’ droplet. After a series of experimental cycles where droplets have been selected based on brightness, the PIs will turn to sequencing to rigorously determine whether there has been a selection effect by comparing the polymer-sequence-space obtained from droplet-selection experiments to the sequence-space of ‘control’ droplets (selected at random; irrespective of UV brightness). If successful, this project would provide the first direct experimental support for theoretical models which suggest that the emergence of cooperating sets of information polymers—and thus the genetic system of modern cells—is a result of selection on the emergent fitness of polymer-rich chemical ecosystems.

Milo Lin, a physicist and Assistant Professor in the Department of Bioinformatics at the University of Texas Southwestern Medical Center, has developed a "mapping” that, when combined with thermodynamic circuit theory, allows one to transform a biochemical network into an equivalent electrical circuit that obeys Ohm’s law. This allows the theorems and powerful quantitative methods of electrical-network-analysis developed over the past century to be applied to biochemical networks. These electrical engineering tools have -for the electronics industry- enabled large-scale system prediction and design through abstraction and modularity as opposed to simulation of all the components and interactions in a given system. Funds from this grant support efforts by Professor Lin to deploy this framework in the analysis of biochemical systems. Lin will begin by systematically mapping a wide variety of biochemical networks found in organisms (regulatory networks, metabolic pathways, molecular motors, etc.) to equivalent electric circuits and then using electrical engineering tools to obtain the simplest circuit of that type. He will then use computer simulations to “evolve” this simple circuit to meet various targets, including determining circuits necessary and sufficient to execute arbitrary computations, circuits that exhibit robustness to input noise, and circuits that exhibit robustness to changes in the fitness landscape.  Lin will then explore the possible existence of a threshold thermodynamic force above which biomolecular computational capacity is dramatically increased, which, if there is a such a threshold, may shed some light on the puzzling observation that living systems overwhelmingly choose nonequilibrium over equilibrium chemistry for computation. If successful, Lin’s project will facilitate our understanding of complex biochemical networks and therefore of how organisms use chemistry to achieve life-sustaining functions. More speculatively, characterizing the computational capacity of a wide range of biochemical networks may provide insights that allow one to delineate living from nonliving matter.

This grant supports research by Chunte (Sam) Peng, an Assistant Professor of Chemistry at Massachusetts Institute of Technology (MIT) and a member of the MIT-Harvard Broad Institute, which will focus on creating novel probes for single particle tracking to quantitatively study the nonequilibrium dynamics of molecular motors in vivo, using these dynamics as a window into the functioning of living systems and far-from-equilibrium physics. Professor Peng proposes to learn about intracellular dynamics by making movies of molecular motors ‘tagged’ with fluorescent probes. The probes—called Up Conversion NanoParticles (UCNPs)—have three features that make them superior to existing fluorescent probes and which suit them for intracellular single particle tracking. First, UCNPs fluoresce at different colors than the light emitted by other cellular components, thus offering superior contrast relative to nearby objects. Second, UCNP fluorescence is spectrally much narrower than that of commonly used fluorescent probes, allowing a larger number of distinct cellular components—each tagged with a different color—to be simultaneously tracked. Third, UCNPs can be used to track a cellular object for much longer (hours or days) than is typical using existing fluorescent probes (a few minutes). Peng will use UCNPs to study the nonequilibrium dynamics of two molecular motors, dynein and kinesin, as they transport ‘cargo’ from place to place within a cell along intracellular protein polymers. The research team will attach probes to cargo and/or motors and then record movies that track probe position as revealed by the UCNP fluorescence. The plan is to begin by quantifying motor dynamics at various points in a motor’s traversal of a cell and then explore why motor behavior varies by gaining an ever-increasing level of detail about the local cellular environment. Particular experimental attention will be paid to how motor efficiency varies in relation to varying cellular conditions, efficiency-affecting interactions between motors and between motors and cargo, and the relation of observed motor efficiency to efficiency constraints predicted by thermodynamic theory. 

Multicellular organisms operate through cell differentiation.  What begins as an initial pool of uniform, isogenic cells eventually develops into a suite of distinct cell types, each performing specialized functions in the organism as a whole. This specialization process can be thought of as a form of information processing . A simplified overall picture of this information processing is that a receptor -for instance at the cell membrane- detects a signal molecule and a signaling network then acts as 'wiring' to relay information to downstream components, which then produce an appropriate cellular response. The signaling network is a chain of biochemical events which link an upstream molecular signal to a downstream 'cis-regulation' system in order to achieve a particular gene-expression pattern that--in this case--specifies cell type.  This grant supports work by Michael Elowitz, Professor of Biology and of Bioengineering at Caltech, to create a synthetic cell fate control system. The system, if successful, will allow one to begin with a pool of genetically identical cells and then -using a small number of 'input' signals- direct various subsets of the pool to differentiate and develop into distinct, predetermined cell types. Elowitz proposes to achieve this goal by using the tools of synthetic biology to develop the two core subsystems briefly sketched above: a 'signaling network'; a complex network of proteins to process chemical 'signals' and relay them downstream and a downstream 'cis-regulation' system that drives expression of a specific gene or set of genes. The signaling network and gene regulation system will be integrated into natural cells to demonstrate cell fate control and differentiation into targeted cell types. If successful, this project will advance our understanding of and control over cellular information processing and provide a foundation for extending synthetic biology into multicellularity.