The information stored in genes plays a huge role in directing the cellular processes underlying life, but stored information alone is inadequate to explain how cells function. Cellular forces and associated motions also play a decisive role in determining whether and when genetic information is expressed because DNA can only be copied when a chromosome is in its decompacted state and because forces and other cellular dynamics drive the transition between compacted and decompacted chromosome states. Forces are also central to the creation and migration of chromosomal density fluctuations, pockets of compaction in a nominally decompacted chromosome region (or of decompaction in a compacted region). These migrating density fluctuations can, in different scenarios, contribute both to biological function and to disfunction. Funds from this grant support Andrew Spakowitz, a Professor of Chemical Engineering and of Materials Science & Engineering at Stanford University, to develop theoretical models that will advance our understanding of how forces/dynamics impact the organization and function of chromosomes in eukaryotic cells. Spakowitz plans to achieve a multi-scale model via a staged progression of model development that describes the coupled chemical/mechanical dynamics starting at the molecular scale, he will then expand to intermediate-scale chromosome dynamics (about a tenth of a chromosome), and end with an exploration of dynamics at the scale of an entire chromosome or group of chromosomes. Forces to be modeled include constraining forces that arise from chemical bond formation between two typically distant segments of a chromosome that happen to come into proximity owing to the wiggling motion of a chromosome in the aqueous environment of a cell and which, in turn, promote chromosome compaction; thermal agitation forces (owing to collisions with water molecules) that drive chromosome decompaction; and the force exerted on DNA (by RNA polymerase) during transcription (reading of genetic information).  if successful, the project will improve our understanding of how coupled mechanical and chemical interactions at the molecular-scale drive the organizational dynamics observed at the much larger length scale of a chromosome or group of chromosomes, thereby providing insight into how forces mediate access to and use of genetically encoded information.

Building a simple form of cellular life from scratch is an ambitious goal, and working towards that goal will advance our understanding of living systems. One promising strategy to achieve this goal is based on the intuition that by avoiding the highly evolved and complex biomolecules found in present day cells, it should be possible to build a (proto)cell that’s much simpler than a natural cell. Professor Katarzyna (Kate) Adamala, an Assistant Professor of Genetics, Cell Biology and Development at the University of Minnesota, takes a different view. She’s open to using any molecules at her disposal to build life from scratch, and her approach to circumventing the complexity of modern cells is -somewhat paradoxically- to dive into molecular complexity. Her thinking is that it should be possible to engineer a complex, multifunctional protein that does what natural cells use several proteins to achieve. If she’s right, then what’s achieved by a complex network of chemical reactions in a natural cell could be achieved by a much simpler set of reactions in a synthetic cell that leverages more complex proteins. This grant supports Professor Adamala’s work aimed at circumventing the complexity of the chemical networks found in present-day cells by engineering more complex proteins. Natural cells are limited to building a tiny fraction of all possible proteins; primarily because they’re restricted to using a small fraction (22) of the known amino acids (~500). Adamala will study what limits which amino acids natural cells use to build proteins, circumvent those limits, and engineer a protein synthesis system -and an associated synthetic cell- that can build a wider range of proteins than can be built by natural cells. Adamala and her team plan to learn about the limitations of natural protein synthesis and expand the chemical diversity of translation via three activities. First, they will evolve the standard ribosome so that it’s capable of building proteins from noncanonical amino acids (ncAAs), amino acids other than the 22 used by natural cells. Second, they will engineer a modified version of a certain protein that’s known to ‘rescue’ failed protein translation. The modified protein will be able to rescue stalled translation involving noncanonical amino acids. Third, the researchers will engineer an RNA translation system that incorporates up to 20 ncAAs. Adamala and her colleagues will use these three products to create a synthetic cell -a liposome vesicle encapsulating “cytoplasm”--that is capable of expanded translation.

An emerging global community of several hundred researchers has started to build cells from scratch by combining small molecules, purified proteins, lipids, and synthetic DNA to assemble simple synthetic cells from scratch. While this community has developed a wide range of ‘modules’ that implement various cellular processes and functions, the fact that these modules are developed independently (in different laboratories) is creating obstacles to progress. For instance, modules are intended to operate within a synthetic cell fluid (cytosol) but their creation across independent laboratories means that they are not developed in a standardized cytosol. This often leads to incompatibilities whereby the conditions that optimize the chemical performance of one module render another either non-functional or poorly performing. This grant to Richard Murray, a Professor of Control & Dynamical Systems and of Bioengineering at Caltech, and Akshay Maheshwari, co-founder and CEO of a company (b.next) “working to democratize synthetic cell engineering”, supports the continued development of Nucleus, a platform that provides a standardized suite of hardware components (chemicals / molecules) and procedures / recipes that can be used to build synthetic cells at varying levels of complexity. Murray and Maheshwari plan to develop three ‘modules’ that expand the technical capabilities of Nucleus, and to enclose the improved Nucleus cytosol within a lipid membrane to produce the first Nucleus synthetic cell. First, they will characterize the Nucleus cytosol performance (yield of transcription and translation) as a function of cytosol molecular composition, with the goal of identifying the molecular composition that best supports simultaneous operation of various modules. The Nucleus cytosol will be characterized across a wide range of various small molecule and protein concentrations, as well as under various conditions of RNA and ribosome abundance. They will also develop tools for implementing modules as ‘DNA constructs’, custom-designed DNA sequences that can be used to synthesize targeted proteins. Murray and Maheshwari will then develop three modules to improve the Nucleus cytosol. The first module aims to increases energy capacity by fostering energy recycling. The existing Nucleus cytosol uses certain energy-resource molecules that when metabolized produce toxins that accumulate and eventually poison the cytosol. The plan is to leverage a chemical pathway that uses a certain enzyme to regenerate the energy resource molecules from the toxins, thereby removing the toxins and replenishing energy resources.  The second module aims to control protein-expression through dynamic (time varying) control of protein abundance.  The existing Nucleus cytosol only allows for protein synthesis, with no ability to reduce protein abundance; a situation that amounts to a limitation on the ability to control the dynamics within a synthetic cell. The team will develop a module that uses a protein enzyme to continuously degrade targeted proteins and thereby provide temporal control over protein abundance. Lastly, the team will develop a ‘membrane-protein module’ and encapsulate the improved cytosol to create a Nucleus synthetic cell. The module will be used for inserting proteins into a synthetic cell lipid membrane; proteins that can transport nutrients into and waste out of a synthetic cell, as well as enable membrane-based cell-cell communication and implementation of various membrane-based molecular sensors. Finally, Murray and Maheshwari will encapsulate the improved membrane-module-enhanced cytosol and reoptimize the performance of the encapsulated cytosol to create a Nucleus synthetic cell. If successful, this project will lead to a novel, open-source platform for building synthetic cells that is accessed and further developed by the global synthetic cell community.

Unraveling how mechanical forces / physical effects contribute to biological function is an underexplored yet important aspect of understanding living systems. This grant provides continuing support to a trio of early-career researchers at Rockefeller University for a series of experiments geared at understanding how mechanical force impacts two important biological functions: the copying of information stored in DNA (transcription) and the coordinated development of a field of cells into a multicellular functional unit (here, skeletal tissue). The proposed transcription research will study how force impacts the dynamics and fidelity of the primary biomolecular machine responsible for transcription, RNA polymerase (RNAP). Forces will be applied to RNAP either using laser tweezers or via collisions between RNAP and various biomolecules that mimic RNAP collisions in live cells. Fluorescence microscopy will capture RNAP dynamics and cryogenic electron microscopy (cryo-EM) will reveal how the molecular-scale structure, and therefore the biochemical activity, of RNAP is modified by collisions. The tissue development research will use cryo-EM along with cellular biology methods that stimulate cell contraction -and thus force propagation- to study changes in molecular architecture that are driven by supracellular (beyond a single cell) force transmission.

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.