Recent improvements in electron microscopy have had a profound impact on cellular biology as they promise to bring a long-held 'holy grail' within reach: the ability to directly visualize nanoscale processes occurring within a cell. This goal—in situ visualization—is important because biological components often change their structure or function when removed from their native cellular environment. This grant supports a project by a team of five principal investigators to build a new type of microscope—a cryo-super-resolution microscope optimized for correlative light- and electron-microscopy (CLEM) that produces 3D snapshots (cryo-electron tomography; cryo-ET)—and to demonstrate the new microscope’s usefulness for the in situ visualization of intracellular structures and processes. Grant funds will support microscope design, construction of the super-resolution microscope, creation of software to run it, and the development of “workflows” defining how to implement CLEM when cryo-ET is the electron-microscopy technique of interest. Additionally, the principal investigators will conduct four demonstration projects in biology that leverage the capacities of the new microscope to obtain in situ structural explanations of a major cellular process. The first project, the clathrin project, seeks to understand force generation during clathrin-mediated endocytosis (CME), the taking-in of matter by invagination of a cell membrane. The second will produce images that improve our understanding of autophagy, the engulfment and degradation of various harmful objects within a cell. The third project seeks to understand the basic mechanisms behind the formation of a primary cilium, a hair-like entity that serves as an important sensory organelle on virtually all animal cells. The fourth project will use microscopy to visualize (polycomb-regulated) changes in chromatin structure during cellular development. The proposed work represents a next-logical-step in the development of electron-microscopy-for-biology and our corresponding ability to study the cell, the most basic unit of life. To help push this emerging technology out to a broad community, information on how to build and use the microscope will be made publicly available and access to the new microscope will be provided to scientists from across the globe through a visitor program.

While we have a handle on the main outline of how life likely evolved on earth, the details remain shaky. It is widely accepted that the essential structures underpinning life on earth—DNA and proteins—originated from simpler substructures, nucleic and amino acids swimming freely in a primordial soup before combining into those complex structures. It’s also widely accepted that nucleic acids paved the way for single-stranded RNA, which doubled-up to become DNA. But just how that sequence of events took place is an area of intense controversy in origin-of-life research. Just how, exactly, did RNA manage to outcompete its rivals and stick around? Charles Carter, an expert in origin-of-life science at the University of North Carolina, Chapel Hill aims bring the debate into the laboratory, exploring the properties and interrelations of RNA and amino acid chains (“peptides”) found in humans. Carter suspects that RNA and peptides coevolved together, a hypothesis that stands in contrast to the current leading theory that highlights RNA as the key molecule. Carter’s hypothesis is grounded in part in the tight bonds RNA and peptides are capable of forming, bonds that require a strong structural similarity that seems unlikely to have happened by chance if they did, in fact, evolve independently. The team will focus on their efforts on 20 RNA-peptide pairs that play an important role in protein synthesis, the critical cell process that uses DNA as a template, to create RNA molecules which, in turn, create proteins, using complex machinery in the cell’s cytoplasm. First, the team will seek to identify ancestral molecules that could have given rise to these contemporary RNA-peptide pairs. Next, they will synthesize copies of those ancestral molecules. Finally, they will use those copies to perform a series of experiments to determine important structural and chemical properties that would be consistent with the RNA-peptide scenario for the origin of life. Answering these questions would not just give us a plausible historical story about how life did emerge on Earth—it would also tell us something more fundamental about how life can emerge, be it on Earth or elsewhere. 

Humans and nature use radically different principles to solve design problems. When building machines, human engineers tend to think in terms of discrete subunits, each with a unique function, coming together to create something greater—the motherboard, processor, graphics card, hard drive, and RAM combine to create a computer, for instance. Nature, however, works with a different set of design principles. While different parts of an organism certainly have unique functions, those functions are often not realized in discrete subunits but are instead distributed throughout the organism. If we look at systems only through our own approach to design, we might fail to understand biological systems and limit our ability to create in interesting ways.  This grant supports Donald Ingber at Harvard University who, together with a talented team of biologists, is exploring one of nature’s ubiquitous designs: hierarchical self-assembly. In Earth biological life, hierarchical self-assembly is achieved through nucleic acid subunits coming together to form intricate strands of DNA which, in turn, go on to guide cell development and differentiation. Grant funds allow the team to explore how aspects of molecular structure contribute to these critical, wide-ranging functions. First, they will use simulations to design different DNA-based molecular scaffoldings. Next, they will build physical copies of these scaffoldings and take measurements to see how the properties compare in reality. Finally, they will grow cells on the different scaffolding combinations to understand how the underlying structure impacts distributed functions throughout the cell. Ultimately, the set of experiments aims to advance our understanding of self-organization across multiple scales—and how variations in underlying DNA structures can impact functions at the cell, tissue, and organism level.

Living systems rely on nanoscale molecular “machines”, many of them proteins, to perform a range of essential tasks such as transporting molecular cargo from place to place within cells, causing muscles to contract, and copying genetic information. Developing the ability to build such machines from scratch is a longstanding goal at the interface of biology, physics, chemistry, and engineering. While researchers are now able to synthesize a wide range of complex protein structures, the molecular machines we're currently able to build pale in comparison those used by living systems. The big difference is that while we've largely mastered the ability to synthesize static nanoscale structures, we don't yet know how to build biomolecular machines, structures capable of the complex mechanical motions that power advanced functionality at the nanoscale. This grant funds a project by a team led by David Baker at the University of Washington to create the first artificial protein machines designed from scratch. The team proposes to build rotary molecular motors consisting of self-assembled axle and ring nanostructures that use chemical and/or light energy to perform mechanical work. Baker will attempt to build a nanoscale rotary motor that will be realized via two primary efforts: design of self-assembling axle & ring nanostructures, and the coupling of relative motion (ring about axle) to the consumption of chemical energy by introduction of catalytic sites at the interface between the axle & ring components. Nanoscale imaging, mass spectrometry, and molecular manipulation will be used to verify the designed structures and their functionality.  If successful, the project has the potential to launch a new field—de novo design of biomolecular machines—whereby different inputs (chemicals, light, electrical potentials) are coupled to molecular systems to enable mechanical motions that provide a new way to manipulate matter on the atomic scale. Success would also represent a vast improvement in our understanding of nature's biomolecular machines and of the cellular biology they facilitate.

Compartments, i.e., discrete spatial regions, are important to life. Cells use them to store molecules for later use and to promote biochemistry by keeping molecules close to one another. Life typically compartmentalizes via walls built from lipid membranes, but biologists have discovered that life also employs a membrane-free approach to compartmentalization. Rather than relying on a wall to demarcate what's inside vs outside of a compartment, life can instead employ phase-transitions, changes in the state of matter (e.g., liquid or solid), to segregate molecules. Envision a gel-like region of closely interacting chemicals within a large liquid-like region of a cell. These phase-change compartments are called biomolecular condensates. They are ubiquitous in living systems and important to cellular function. Condensates have been identified that sequester damaged proteins and orchestrate chromosome separation during cell division, and they've also been implicated in human diseases such as Alzheimer’s. This grant funds work by a team led by Rebecca Shulman at Johns Hopkins University who are attempting to understand and ultimately control condensates as a step towards an improved understanding of compartmentalization that could shed light on the matter-to-life transition. Condensates appear well-positioned for this purpose. On the one hand they share many core functions with cells. Condensates naturally form, grow, dissolve, fuse, and divide. On the other, they are vastly simpler, biomechanically, than cells themselves, allowing researchers to investigate these core activities without the complicating internal structure present in natural cells. The work will focus on nucleic acid-based condensates, condensates where the nucleic acid polymers DNA and RNA are the functional elements that condense or otherwise control the phase transition between a condensate and its exterior. The team's approach is to design, build, and characterize nucleic acid condensates. They'll design synthetic DNA templates that produce RNA molecules with the capacity for spontaneous phase separation, testing hypotheses about how aspects of polymer design influence the condensation process. They'll use optical microscopes and other tools to achieve high-throughput characterization that leads to phase diagrams that summarize condensate properties under varying conditions (temperature, different condensing macromolecules, various solution-phase buffer-molecules). They'll also develop molecular signals that cause distinct condensates to mix or demix with one another, or which cause one type of condensate to expel molecules that signal another condensate to perform some function. These studies will provide insights into how condensates can be used to transport "cargo," chemicals that are not essential to the condensation itself but are instead stored or transported by the condensate. If successful, the project could form the foundation of a new discipline of condensate engineering, one that could open new routes to chemical synthesis, advance our understanding of natural cells, lead to new types of intracellular organelles, and provide insights into how matter transitions to life.

Nonequilibrium dynamics is a thriving sub-field within physics that seeks to identify the principles underlying complex spatiotemporal patterns that arise in far from equilibrium systems. Living biological organisms are one important subclass of systems far from equilibrium, yet, due to their complexity and variety, they have remained relatively understudied by theorists and experimentalists alike. As such, significant questions exists both about the extent to which methods of nonequilibrium dynamics can be used to identify laws governing pattern formation and regularities in biological organisms. Funds from this grant support Jeff Gore, Nikta Fakhri, and Jörn Dunkel of MIT in a series of projects that will begin to examine whether and how nonequilibrium dynamics might be used as an organizing framework for understanding how ordered biological phenomena arise and evolve across a variety of scales. Topics to be explored by the team include the role of topology and topological defects in triggering order-enhancing processes in starfish cells; the assembly of ordered structures of colloidal molecules by motile bacteria; and how spatial distribution affects the evolution and ecology of microbe populations. In addition to knowledge gained, the project will involve the development and deployment of new imaging and theoretical analysis tools, expanding the available methods for the thermodynamic study of biological systems.

Natural cells routinely use shape as a vector for acquiring and disseminating information about their environment.  Detecting the shape of a cell they have encountered can impart important information about their neighbor, and thus inform what sort of response would be most adaptive.  The underlying mechanisms that allow organisms to process this topological information, however, are not well understood.  This grant funds a multi-disciplinary a team led by Ronit Freeman at the University of North Carolina at Chapel Hill to better understand these mechanisms by attempting to create an artificial cell-like entity that mimics natural cells’ ability to detect shape.  Grant funds will support three interrelated research efforts. We know that cells use proteins to detect the shape of fellow cells, but the proteins that perform this function can be 10 to 100 times smaller than the cells they are measuring.  How these natural systems bridge that length gap is poorly understood.  Using advanced atomic scale microscopy, Freeman’s team will observe the behavior and structure of these shape-detecting proteins and attempt to reverse engineer synthetic versions that could perform similar functions in a synthetic cell. A second effort will focus on signal transduction, the process of converting shape data collected at the cell membrane into physical and biochemical signals that can be passed to a cell’s interior.  The research team will attempt to create synthetic pathways that mimic signal transduction mechanisms thought to operate in natural cells, allowing them to use detected topological information to effect changes in behavior of the synthetic cell itself.  Third, the team will use advanced techniques to model how topological information can spread through communities of cells, affecting the behavior or characteristics of entire cell collectives.  Such modeling has, to date, mainly confined itself to the chemical aspects of cellular communication. Freeman and her team will expand these efforts, incorporating physical variables such as the flow of mass and momentum, membrane elasticity, flow across interfaces, and cell deformation into their model.

A Cornell University team led by Paul McEuen has developed a fully artificial platform with some remarkable capabilities.  The platform's basic building blocks—magnetic digital polymers—are small panels (a few microns) with magnetic data lithographically patterned on their faces and sides. These data specify via magnetic forces how the panels interact with one another.  This ingeniously allows the panels to mimic the chemistry of biological molecules.  In biological molecules, the atoms that make up a molecule determine which other molecules it can chemically bond with and how strong such a bond, once formed, is.  By altering the data pattered on the faces of the digital polymers, McEuen and his team can replicate these features, with some polymers bonding selectively with others, just like biological molecules do.  What’s more, because the physics of magnetism is well-understood, the behavior of McEuen’s magnetic polymers should be relatively simply, at least in theory, to model and predict. This grant funds an effort by McEuen and his research team to attempt to use magnetic digital polymers to mimic two important features of biological life: reproduction and metabolism. To demonstrate "reproduction" McEuen and his team will begin by developing what they call Magnetic DNA, a digital magnetic polymer capable of replicating itself. Reproduction will be demonstrated by programming in appropriate magnetic interactions to create information strands (polymer patterns) that self-replicate under cyclic application of “agitation” via an external magnetic field, acoustic waves, and/or thermal excitation. To demonstrate "metabolism," the team will use a variety of strategies to create a magnetic polymer version of an enzyme, an entity that can modify the replicating unit. This will involve using magnetic and mechanical forces to cut linear polymer chains at specified locations, in analogy to how the CRISPER-Cas9 protein cuts DNA.

Speculation about the existence of other life in the universe has become invigorated in recent decades by an explosion in the discovery of extrasolar planets.  The numbers tell the story. There were about 50 known exoplanets in year 2000, about 500 known by 2010, and we're approaching 5,000 today.  Powerful telescopes can reveal information about the chemical composition of the atmospheres of these far away planets.  Can they also tell us if there is life there?  They could if the atmospheres of planets with biospheres differed systematically from the atmospheres of lifeless planets, Knowing that, however, would require knowing what the atmosphere of a lifeless planet looks like, and how life might change it. This grant funds an effort led by Anan Shahar at the Carnegie Institution for Science to determine the abiotic atmospheric baseline of the most common planets in our galaxy, sub-Neptune and rocky planets. With the abiotic baseline known, scientists can then consider how Earth-like life might change a planet's atmosphere and in this way tackle the question of whether or not it's possible to determine signatures of life by studying exoplanet atmospheres The research team will pursue an interdisciplinary, holistic approach that combines solid-planet expertise with atmospheric expertise in order to understand the baseline abiotic atmosphere of a planet and how it evolves over the planetary lifecycle.  What’s called a planet’s primary atmosphere is formed early in a planet’s  formation, as the planet coalesces from matter orbiting a local star.  This atmosphere then evolves into a secondary atmosphere, one shaped both by geologic processes on the planet itself (volcanism, magma oceans, outgassing, the weathering of the planet’s surface) and by external forces like comet impacts.  The research team will attempt to model and the analyze the effects of these forces, shedding light on how the atmospheres on lifeless planets are likely to evolve across the planetary lifecycle.