The encoded, heritable information that plays a large role in determining an organism’s traits is known as the organism’s genotype. In natural organisms, genotypes are specified by an organism’s DNA. By contrast, an organism’s observable traits (its phenotype) are determined by proteins produced from the information encoded in DNA. All known forms of life use this dual-molecule structure: distinct molecules for genotype vs. phenotype. Separating the molecular basis of genotype and phenotype, however, is thought to be a highly evolved approach to leveraging and expressing information, and it’s conjectured that the earliest forms of life employed a simpler system whereby one type of molecule specified both the organism’s genotype and phenotype. This grant supports work led by Job Boekhoven, a Professor of Supramolecular Chemistry at the Technical University of Munich, to construct a purely synthetic cellular system (one composed of non-biological molecules) that is at once both self-replicating and capable of evolution, yet uses a single molecule for both genotype and phenotype. Boekhoven and his team aim to develop a chemical system of self-replicating molecules that encodes heritable information (i.e. develop a genotype) and then incorporate these ‘genes’ into artificial vesicles that can divide.Some of these self-replicating molecules will be engineered to have measurable effects on the larger vesicle, affecting characteristics of the vesicle membrane and cytoplasm, thus coupling the cell’s genotype with its phenotype. This would pave the way for further experimentation, not funded by this grant, that would demonstrate that these phenotypic expressions could be adaptive or maladaptive in various environments, and thus form the basis for evolutionary selection.

Funds from this grant support a project by Michael Murrell and Enrique De La Cruz, Professors of Biomedical Engineering & Physics, and Biophysics & Biochemistry respectively at Yale, to explore the role that thermodynamics plays in driving cell division. Murrell and De La Cruz hypothesize that the behavior of a cell’s actomyosin cytoskeleton, a ring-shaped network of filaments, motor proteins and connectors that contract to pinch a cell in two, is shaped by thermodynamic principles, and that contraction of the network can be explained by reference to the fact that contracting and dividing would move the skeletal network into a more energetically favorable state. Murrell and De La Cruz will leverage a ‘reconstituted’ actomyosin cytoskeletal network comprised of purified and synthesized cell components which will allow them to study the system’s properties and behaviors outside the complicating environment of a cell. The team will develop new measurement techniques to measure and quantify key thermodynamic parameters of this system: how much entropy is produced, the energy input to the system, the energy output as mechanical work, and the energy lost as heat and validate these measurements to ensure they yield consistent findings (i.e. no missing energy). The team will then apply these techniques to various configurations of the system, measuring how efficiency varies with system structure, composition, and dynamics. They will then insert the artificial network into a cell-sized lipid membranes to measure how these thermodynamic properties vary during the various stages of an actual process of membrane division. This will allow them to test whether ring formation and contraction in a cell-like geometry is energetically favorable compared to a non-contracting steady state.  The proposed experiments will quantify the thermodynamics of the actomyosin cytoskeletal system at the heart of cell division, and in doing so make an important contribution to an emerging body of knowledge about cellular thermodynamics.

Funds from this grant support research by Patrick Oakes and Jordan Beach, both Professors in the Department of Cell and Molecular Physiology at Loyola University Chicago, to better understand how a cell’s energy supply is linked to the mechanical forces it generates—an important gap in our understanding of how cells regulate energy resources to coordinate basic functions such as movement, division, and changes in shape. Oakes, Beach, and their team will use Sloan funding to measure how cellular ATP levels (the primary form of usable energy in cells) relate to force generation by the actomyosin cytoskeleton, an intracellular protein network that drives contraction and is a key player in cell division and motion. The work will be done using live cells, with experiments that both increase and decrease ATP  availability to see how the actomyosin skeleton responds, as well as with experiments that stimulate cytoskeletal activity to see how cellular ATP levels and other major energy-consuming processes respond. The project will also examine these relationships at finer spatial scales inside cells. Using imaging-based metabolic sensors and force-measurement methods, the research team will map and quantify where ATP is higher or lower inside the cell and compare those patterns with where contractile forces are generated. They will also field a series of experiments where ATP levels are manipulated in localized regions while observing the behavior of the corresponding section of the cytoskeletal network. If successful, the project will produce quantitative measurements describing how cellular energy availability and mechanical force generation influence one another at both whole-cell and subcellular scales, along with datasets and analysis that can help clarify how cells regulate mechanical behavior.

This grant supports experiments to assess whether “xenobiotic nucleic acids” (XNAs)—DNA/RNA-like polymers not found in nature—could serve as alternative carriers of genetic information, with implications for understanding possible early-life chemistries and for building simplified synthetic cells. A team led by Lijun Zhou at the University of Pennsylvania will use Sloan funding to characterize how efficiently and how accurately a specific class of XNA polymers (NP-DNA and NP-RNA) can be copied from a template without the use of enzymes. The work will measure copying speed and error rates across a diverse range of XNA sequences and varying environmental conditions (such as pH, temperature, and ion concentrations). The team will also investigate how the addition of reactivity-enhancing biomolecules affects copying speed and fidelity and whether and how genetic information could be transferred between the two types of polymers. In addition, the project team will run laboratory evolution experiments to determine whether these XNAs can undergo Darwinian evolution to expand their functionality, focusing on evolving XNA sequences that can catalyze useful reactions, such as joining short XNA strands together.

This grant provides support for the Build-A-Cell research coordination network, a collaborative, international community of more than 100 scientists drawn from about 100 labs across the globe whose work focuses on building synthetic cells. The Build-A-Cell network will use Sloan grant funding to run two in-person workshops each year and to support ongoing working groups that collaborate between workshops. The workshops are designed as hands-on working meetings that bring participants together to exchange methods, compare results, identify shared technical challenges, and coordinate community-led projects. The working groups will pursue targeted objectives across areas such as modeling, integration of cell components, education and outreach, biosafety and security, international engagement and policy, and biomanufacturing. Sloan funds will be used to support the logistics needed to sustain these activities, including travel support for workshop participation and partial support for a coordinator who will organize meetings and help keep working groups moving.