Spiny pufferfishes, e.g., porcupinefish, defend themselves by transforming from a streamlined torpedo to a large, prickly sphere. This dramatic yet reversible shape morphing is achieved by highly mineralized hard spines anchored to a very extensible soft skin that stretches up to 40% as the body inflates. The armored skin is activated by body inflation: as the skin stretches, the sharp spines rotate into an erect position, rigidly pointing outward as a visual warning and mechanical defense. Interfacing between hard and soft tissues is ubiquitous in nature like bone and tendon, but it is very unusual for such an interface to embrace extensive, lifelong distortions while maintaining the material integrity. Remarkably, the skin-spine interface of pufferfish does exactly this function, dramatically morphing both skin and spines with every inflation-deflation cycle and using this transformation as a defense mechanism. How do puffers assemble this unlikely combination of extensible and rigid materials into a repeatedly stretched interface—without tearing themselves apart? How does the drastic skin extension drive and guide spine rotation? How do properties of soft skin and hard spine reciprocally shape dynamic movements? In this project, we integrate our expertise in mechanics, biology, and robotics to provide unprecedented insights into the multi-scale architecture and complex function of the porcupinefish’s armored skin. We will systematically investigate the dynamics of the body expansion, skin stretching, and spine erection and adapt the deployed structural and architectural strategies to create bio-inspired functional interfaces. The micromechanical experiments will reveal the secret to the integrity and performance of the interface of materials with contrasting properties. The musculoskeletal measurements, both on live animals and fresh tissues, shed light on the kinematics and functional morphology of the armored skin of pufferfish. The soft robotics model presents a cyber-physical twin for pufferfish capable of reproducing reactive behaviors consistent with its morphing behavior to verify and generate biological hypotheses. Our collaborative project unravels how the interfacial morphology of pufferfish skin encodes complex shape-morphing functions transferable to adaptive architectures for biomedical, surveillance, and environmental monitoring application.

Large polysaccharides are widely found in nature. Arguably one of the most complex and biologically diverse groups of polysaccharides are the glycosaminoglycans (GAGs) that despite a relatively simple disaccharide repeating backbone attain enormous structural variation through decoration by sulfate groups. While many important and diverse bioactivities assigned to GAGs are believed to be directed by distinct patterns of sulfation along the large GAG chains, direct structural identification of these motifs remains largely unexplored due to analytic barriers. Therefore, our aim is to marry emerging technology advances to make a quantum leap in glycosciences with atomic-level sequencing and structural determination of polysaccharides at the single-molecule level. We hypothesize that precise genetic engineering of glycosylation in cells (RLM’s team at UCPH) will enable us to produce sufficiently homogeneous GAG chains for direct sequencing analysis by single-molecule imaging using electrospray ion-beam deposition and scanning tunneling microscopy (KA’s team at MPI). We focus on GAGs that through complex sulfation patterns orchestrated by 30 distinct sulfotransferases produce recognition landing paths - sulfation codes - for a myriad of GAG-binding proteins that serve essential functions in all metazoans. We will produce these GAGs from the library of cells, and they will be structurally characterized by imaging them one-molecule-at-a-time using scanning tunnelling microscopy to provide the visual form and shape. The images corroborated by ab initio calculations at the level of Density Functional Theory (DFT) will reveal structural GAG motifs that give rise to distinct bioactivities. We believe this interdisciplinary strategy will enable us to dissect the bioactive cues embedded in the long GAG chains. Moreover, we believe that the strategy of combining genetic glycoengineering to reduce the complexity and heterogeneity of GAGs combined with single-molecule imaging will lead to a major breakthrough in glycosciences, which will more generally open up opportunities for direct analysis of other types of polysaccharides, including bacterial lipopolysaccharides, plant polysaccharides such as chitosan, as well as more complex glycoconjugates such as proteoglycans and glycoproteins.

All life on Earth depends on proteins, now composed of a quasi-universal alphabet of 20 amino acids. The alphabet has been fixed in the Central Dogma for eons of evolutionary history, creating the impression that it is somehow “optimal” for folding sequences into complex structures and performing biochemical functions. The possibility however that the canonical amino acid alphabet is a “frozen accident” (to quote Francis Crick) has yet to be critically examined because whether alternative amino acids would lead to “bad” alphabets has not been empirically tested. Here, we propose to design “xeno-alphabets” composed primarily of non-canonical amino acids, and then to synthesize peptide libraries “written” in these new chemical languages. Our goal is to test whether alternative life (or evolutionary paths) could have produced successful proteins from entirely different building blocks and to learn about the chemical logic of the set found within life’s genetic code. As the number of possible xeno-alphabets is infinite, chemoinformatic approaches will be used to propose representative xeno-alphabets that vary in their chemical distance from canonical and capture different regions of physicochemical property space. After designing such alphabets in silico, we will synthesize peptides composed of these letters using solid phase peptide synthesis (which will allow us to focus on evaluating the properties of the peptides, such as the ability to fold or bind with other biomolecules) or using wet-dry cycling (which will focus on the capacity of the building blocks to form peptides autonomously under prebiotically-relevant conditions). In characterizing these peptide libraries, our principal aim will be to compare the structure-forming and functional potential of the alphabets (using a range of biophysical and biological methods) with the canonical set, to answer: How large is the chemical space that could support life as we know it (or don’t know it)? We, therefore, propose a research program by which unique computational tools will be adapted to design xeno-alphabets, whose peptides will be tested with laboratory techniques – some of which draw from our previous experience but most require significant methodological innovation, including next-generation peptide sequencing.

Defects in microglia, the resident immune cells of the brain, have been implicated in neurological diseases, including Alzheimer’s, Parkinson’s, and multiple sclerosis. Microglia perform diverse functions, such as developmental synapse elimination, immune surveillance, and phagocytosis of pathogens and cellular debris. As highly ramified cells, microglia spatially regulate locally branched domains. In response to local signals, microglia can rapidly switch their shape from highly ramified to amoeboid. How this dramatic morphological change is achieved remains unclear. Recent transcriptome profiling of microglial processes uncovered a subset of local mRNAs encoding cytoskeletal protein. This suggests that mRNA transport and local translation may regulate microglia morphology. Though both of these mechanisms are well understood in neurons, they have not been studied extensively in microglia. In addition, few tools are available to manipulate mRNA transport and local translation with spatial precision. We will capitalize on our expertise in glial cell biology (Fu GLIA Lab), regulatory RNA structure, translation and therapeutics (Leppek RNA Lab), protein biochemistry and liquid condensates (Milovanovic CONDENSATE Lab), and synthetic organic chemistry (Broere SYNTHESIS Lab). We hypothesize that manipulating mRNA granules can lead to local changes in translation and morphology. We aim to: (i) identify key local mRNAs that affect microglial function using primary microglia cultures; (ii) characterize the mesoscale assembly of mRNAs and RNA-binding proteins (RBPs) inside condensates; (iii) leverage mRNA structure-function to design and develop a photoswitchable bio-active RNA tool (SMARTswitch); (iv) validate that SMARTswitch can manipulate local translation in microglia. Collectively, we will provide innovative synthetic RNA-based approaches to modulate local cellular functions and reprogram microglial responses.

Genome and organelle sizes vary with cell volume following scaling laws that are thought to reflect physical and molecular constraints on life. Despite cell size spans over several orders of magnitude across the tree of life, individual species are able to maintain a characteristic, narrow range of volumes, following a handful of homeostatic principles. To cover a large range of sizes, scaling patterns were derived by comparing distantly related species, highlighting their conservation but limiting causal inference. On the other hand, much of the work on the mechanisms that maintain size homeostasis was done in single species, which typically cover cell size ranges that are too narrow to investigate scaling patterns. The molecular and physical links that govern size scaling patterns and homeostasis principles, and how they evolve to produce the variability we observe in nature have remained elusive. To overcome previous limitations, we will evolve and engineer a panel of much larger/smaller Saccharomyces cerevisiae cells, aiming at finding the physical or evolutionary limits for this species. We will track genomic and cellular changes associated with the altered size, identify causative mutations, and dissect the mechanisms they act on. Single cell measurements in a genetically amenable species over an unprecedented range of sizes will allow us to test the robustness of homeostasis principles and scaling laws in cell biology, and observe how evolution can alter physical constraints on cell, genome and organelle size.

Associations between organisms and viruses are complex and encompass everything from beneficial to parasitic relationships. Here we will map the molecular mechanisms that underlie the interactions between crenarchaea, their DPANN archaeal symbionts and their viruses. Archaea are ubiquitous microorganisms in the environment, but the number of isolated species is modest and their cell biology remains underexplored. They are regarded direct ancestors of eukaryotes and important for understanding the origin and evolution of life. Current knowledge of microorganism–virus interactions relies primarily on laboratory studies of one virus and one cell. Yet, based on culture independent approaches, the fraction of microbial species that is involved in a symbiotic relationship is estimated to be considerable, although most are unculturable. Symbiotic microorganisms sustain relationships with another organism (called the host) over a considerable fraction of the life of the host. In addition, this relationship is beneficial for at least one of the organisms. Since symbionts often lack anti-viral defence systems, it has been hypothesized that symbionts can protect their host-cells from viral infection, by serving as decoy. However, empirical relevance of this hypothesis has not yet been tested. Recently multiple fast-growing symbiotic archaea belonging to the phylum DPANN were isolated by HS, which now offers the exciting possibility to unravel molecular mechanisms underlying viral infection in a symbiotic system. The aerobic fast-growth allows us to apply to this system recently developed imaging techniques. We will combine virology, microbiology, microscopy and structural biology with mathematical modelling to connect the microscopic properties of the individual cells with the emergent behavior at the population level. This will allow us to assess if symbiotic DPANN serve as viral decoys. Viruses of DPANN archaea are not studied, even though these archaea are widespread. With this project, we will generate unprecedented detail of their infection cycles, structure and the biology of their DPANN hosts, which will lay foundations for a novel field of research. This project will be one of the first to study viral infection in a symbiotic system, and thus provide essential insights into the impact of viruses on symbiotic organisms, which are so widespread in nature.

Most gaseous oxygen (O2) on Earth is produced via oxygenic photosynthesis. However, new evidence indicates that O2 is also produced in permanently dark ecosystems. This so-called “dark oxygen production” (DOP) can proceed via abiotic chemical reactions or via microbial dismutation of chlorite and nitric oxide—metabolisms fundamentally different from photosynthesis. Recent work suggests that microbial DOP is widespread in groundwater ecosystems, yet definitive evidence is lacking to date. Our team has the abilities, instruments, and expertise to provide such evidence and elucidate the origins, processes, and production rates of groundwater O2. We hypothesize that microbial DOP is a globally relevant process in groundwater ecosystems today, and that groundwater aquifers represent model systems to study O2 production and consumption in the deep geologic past, as well as in subsurface ecosystems of other celestial bodies. We propose an international, multi-disciplinary project to provide comprehensive insights into the geochemistry, microbiology, and ecology of DOP. Our combined expertise leverages three key technological advances: (1) triple- and clumped-oxygen isotope analyses, which can unambiguously link O2 to biological DOP origins (led by PI Hemingway), (2) determination of DOP rates and fluxes at unprecedented resolution using nanomolar O2 concentration measurements (led by PI Kraft), and (3) high-throughput analyses of O2-producing enzymes and pathways present in complex communities using genome sequencing and protein mass spectrometry (led by PI Ruff). We will apply these techniques to a sample set from diverse aquifers spanning a broad range of ecosystems in Canada, Finland, Germany, Switzerland, and the USA. We expect several key outcomes. First, we will determine the first ever triple- and clumped-oxygen isotope signatures of DOP by different biotic and abiotic sources, values that are needed to decipher natural signals. Second, we will use stable isotope-labelling to determine rates of microbial DOP in groundwaters, critical measurements that are lacking to date. Finally, we will conduct the first global survey of key microbial lineages and genes to understand the diversity, abundance, and activity of microbes involved in DOP. These insights are crucial to understand groundwater aquifers, which represent Earth’s largest source of drinking water.

Understanding how infants acquire the meaning of words is fundamental to the study of the human mind. Infants master a considerable vocabulary by year 2, yet, learning word meanings is not a trivial task: labels heard can map on many referents in a complex world. Infants must thus constantly aggregate evidence in such ambiguous contexts. Behavioral methods fall short of tracking semantic processing during learning. Therefore, this process, including infant strategies (whether they put their bets on one candidate to reject/confirm it or slowly accumulate evidence) is still poorly understood. To go further, pediatric tools that can peer into the brain networks supporting word-to-meaning mapping in real time are required. Functional ultrasound (fUS) enables noninvasive and dynamic mapping of brain activity in newborns with little restriction. Here, we will use cutting-edge fUS capabilities to investigate neural correlates of representational changes during word learning in 12 months old infants. We will rely on advanced semantic modeling techniques to decode neural correlates of word representations detected by fUS, track such representational changes in the course of learning and unravel infants’ learning strategies.

Single-celled marine algae produce half of Earth’s atmospheric oxygen and are the trophic entry point for life in the ocean. A suite of animal-like ion channels has recently been discovered in members of the red-lineage algae, and has been shown to underlie electrical excitability – the same phenomenon that drives a wide range of sophisticated dynamic behaviors in humans. However, in algae, the full mechanistic basis of this dynamic behavior and its widespread physiological implications remain poorly understood. In this study we aim to determine how ion channels work in concert to produce electrical signals in the red-lineage algae and how these ion fluxes impact the chemistry of the cell. Armed with a quantitative understanding and an ability to simulate electrical behavior we will ask the question: what dynamic behaviors are induced by this excitability? We hypothesize that excitability serves vital roles in marine phytoplankton, including regulating calcification, gene expression, interactions with light, and cell-to-cell communication. We propose that only by understanding membrane excitability within a whole-cell context will we be able to determine how the cellular physiology of unicellular algae results in a wide range of poorly understood phenomena, from exquisite control over intracellular biomineralization, to aspects of population growth affecting global nutrient and carbon cycles. We will take a multidisciplinary approach, integrating mathematical modeling with electrophysiological experiments, pharmacological manipulation, and biochemical approaches, to characterize and explore excitability in two model species of marine algae. The work will be physically split between the electrophysiology lab of McClenaghan at Rutgers University, and the computational microbiology and algal growth laboratory of McClelland at UCL, but will comprise a closely integrated program of work leveraging our contrasting expertise. Our proposed project has the potential to unearth undiscovered behaviors in these organisms, with major ecological, climatic and biotechnological implications.