Exocytosis is a conserved vesicle trafficking pathway responsible for the delivery of biomolecules to the cell surface and to the extracellular media. While regulated exocytosis is present only in secretory cells (i.e. neurotransmission or hormone release), constitutive exocytosis is generally present in all eukaryotes, where it regulates cell growth, cell progression, cell polarity and it is involved in multiple human pathologies including cancer and neurological disorders. Our goal is to time-resolve the molecular mechanism of constitutive exocytosis (i.e. exocytosis). Understanding complex and multi-step processes such as the exocytosis requires a complete overview of the structure-function relationships and the dynamics of the protein machinery involved. Exocytosis is regulated by an extensive network of macromolecules organized in protein complexes, regulatory proteins and membranes with specific compositions. Despite it has been long studied, the complexity and dynamism of the mechanism of exocytosis could not be reconstituted in vitro. This and other technical constraints limited the characterization of the cellular machinery in charge of performing this function. To investigate this further, we have gathered an international team of scientists to resolve the global mechanism controlling exocytosis through the concerted application of advanced imaging techniques. The team includes experts in biology, mathematics and engineering of instrumentation for microscopy. We intend to decipher the molecular mechanism of exocytosis by addressing the system with all of its complexity, including its topology, dynamics and function. We will combine live-cell imaging, the development of imaging instrumentation and advanced mathematical computation to build an enhanced setup capable of reconstructing the cellular machinery beyond current technical constraints. Together we will embark on a comprehensive approach to solve the long-standing question of how the cell is capable to coordinate such a massive and dynamic protein machinery in charge of driving exocytosis.

Despite the central role operons play in prokaryotic genome organization, their origin, evolution, function and unique characteristics are not well-understood. The primary difference between operons and the comprehensively-studied monocistronic mRNAs is that operonic cistrons frequently overlap with one another. These overlaps mandate that the translation initiation of a distal cistron must occur within the coding region of the upstream proximal cistron. This process does not allow for independent translation initiations, a fact inadequately explained by the current paradigm for translation initiation, the 30S binding mode, as it necessitates constant interference between elongating and initiating ribosomes. Recently, a new mode of translation-initiation, the 70S scanning mode, was experimentally observed, and it offers a challenge to the existing paradigm. However, it remains unknown which initiation mode occurs at each cistron, nor how to perform measurements that differentiate between the two modes and capture their genomic distribution inside a living cell. Moreover, it is not known what processes are involved in the ribosomal initiation mode-decision or its mechanism of regulation. Herein, we propose an experimental framework based on ribosomal titration assays followed by ribosome profiling, which will enable us to identify the genome-wide distribution of initiation modes, and measure and map their ribosome-mRNA affinity landscape. Using this data, we will study and characterize the mechanisms that govern the mode of initiation, and build synthetic operons to validate our findings and enable precise prediction and utilization of the operon unique characteristics.

The mitotic spindle is a dynamical apparatus consisting of microtubules (MTs) and associated proteins. Mitotic spindle bipolarity is vital for faithful separation of chromosomes during cell division. The bipolar spindle architecture can be conceptualized by a nematic network of antiparallel MTs at the spindle center and, in many cell types, polar (or astral) MT arrays radiating from the spindle poles. Although most spindle components are known, how they work together when spindles self-organize it is still not clear. It would be illuminating to understand what is the critical minimal set of activities that is necessary and sufficient to form a spindle-like active MT network. This project addresses this question by exploring the morphogenetic potential of active MT networks in 3D space using computer simulations. The project will explore the conditions allowing the coexistence of nematic and polar MT network states as observed in spindles. We will investigate networks organized by antagonistic motors, passive crosslinkers, and regulators of MT nucleation and MT stability. We will study how spatial confinement affects active network organization. We will determine a predictor of network topology based on the biophysical parameters of the underlying components. The computational work will be critically informed and validated by in-vitro reconstitution experiments and theoretical understanding will guide the design of in-vitro reconstitutions. The extracted design principles for self-organizing minimal systems will then form the basis for understanding more diverse and complex spindle networks in living cells.

A biological neural network is formed by living neurons with massive communication links between them. Neurons communicate by delivering electrical signals through insulated axonal fibers, providing the communication speed of up to 100 m/s. Although this biological neural network enables powerful human computation, the more powerful and efficient computation can be made if the neural network equips a faster mode of communication. Inspired by the revolution in the modern communication industry from electrical cables to optical fibers, we aim to explore whether a living neural network can be engineered to communicate with optical signals. Compared to electrical signals, optical communication is faster by orders-of-magnitude and selective by wavelength, which could significantly improve both communication speed and bandwidth. Specifically, we will first construct light-emitting neurons using a ‘cell laser’ technique and light-receiving neurons constructed by optogenetics. Based on these living optical elements, we will develop optical logical circuits on a chip for logical computational operations. We will further incorporate the time-varying property of living neurons by using the light-controlled expressions of photoactive proteins. To successfully achieve the research goal, we have assembled a multidisciplinary team of a physicist (Dr. Humar) for microlasing and optical configuration, a biomedical engineer (Dr. Choi) for neural engineering, and an electrical engineer (Dr. Im) for on-chip integration and analysis. Successful completion of this project will bring a new concept of optically communicating neural circuits that could realize the fastest communication mode in biology.

The experience of traumatic, life-threatening events gives rise to some of the most enduring forms of fear memories, which can degenerate into a devastating pathological state known as post-traumatic stress disorder (PTSD). Yet, surprisingly little is known about how long-lasting memory are formed and stored. Likewise, the neural and molecular mechanisms of how to best overcome remote fear memories remain unknown. The aim of this project is to identify the transcriptional and epigenetic changes occurring at a single cell level during remote fear memory attenuation, and to validate their functional relevance. To this end, I will use a mouse line in which the promoter of a neuronal activity marker (c-Fos) drives a tetracycline inducible system that allows for the identification of the neuronal subpopulation underlying fear memory attenuation. FACS cell sorting coupled with single cell RNA-seq, ChIP-seq and ATAC-seq will capture the genome-wide changes in gene expression and chromatin organization occurring within the tagged subpopulation of cells. Finally, spatiotemporally controlled CRISPR approaches will be used to dissect what gene-specific alterations, either transcriptional or epigenetic, are responsible for remote fear attenuation. I anticipate that this proposal has the groundbreaking potential to uncover the molecular mechanisms behind the overcoming of remote fear memories, providing precious insights for the treatment of PTSD. In extension, showing how the modulation of epigenetic marks in a cell type and locus-specific manner can impact neuronal function and memory capacity will represent a major step forward for the entire field of molecular neuroscience.

Our objective is to elucidate the mechanism, from subcellular to global scale, underlying the interdependence of plants and climate during Earth's current climate transition. This century, rising CO2 will force global temperatures to increase, pushing some vegetation systems into heat stress and potentially physiological collapse, irrespective of rainfall. Indeed, the most vulnerable regions are humid, high rainfall zones where 'wet-bulb' temperature (the lowest temperature that an evaporating object or organism can cool to) will rise to levels known to induce physiological stress and cellular death. Plants crucially affect climate through stomatal regulation of transpiration, which influences partitioning of net solar radiation into heating and evaporative cooling actions at the land surface. Plant vulnerability to heat stress can diminish this role and promote further climate warming, but little is known of the physiological and molecular mechanisms of stomatal regulation under heat stress, or of the combined physiological and climatic conditions that generate lethal leaf temperatures. This limits our capacity to predict and prepare for the long-term effects of global warming. This project will unlock mechanistic information from plant molecular genetics and heat stress physiology to formulate new theory describing stomatal regulation under temperature extremes, and apply this in an Earth system modeling framework to develop tools for predicting and adapting plant-atmosphere dynamics. Our central hypothesis is that previously unrealized breakdown of the stomatal regulation mechanism under heat stress, when expressed at the landscape scale, will add a significant and as yet unforeseen enhancing feedback to climate change this century. To test this, we will undertake the first coordinated integration and scaling of the stomatal control mechanism responding to heat under high CO2, from molecular signaling through to global fluxes, to quantify the full effects of plant heat stress on regional and global climate. Genetic and physiological methods will be used to develop and validate a new leaf-scale model of stomatal regulation at high temperature. This will be implemented in Earth system simulations to re-evaluate end of century climate scenarios in the most heat-vulnerable regions using improved surface energy partitioning calculations.

Biosensors are used extensively to transduce critical biological parameters such as biomarker concentrations into measurable signals. However, current biosensors are poorly suited for studies that benefit from long observation time or live hosts, especially those involving complex biological processes such as metabolism and neural signalling. This can be due to either low in vivo stability, limited sensitivity, short monitoring times, or high invasiveness. To overcome these limitations, I propose the design of a novel class of biosensor combining the specificity and stability of protein-based biosensors with the minimal invasiveness and long monitoring times of electrochemical sensing. These biosensors will be based on periplasmic binding protein (PPBP) scaffolds, which convert binding of an analyte into a conformational change. As local analyte concentrations fluctuate, this conformational change will be harnessed to move a redox sensitive probe, such as a ferredoxin, in relation to the surface of an electrode on which the sensor will be immobilized. As probe proximity will in turn be detectable through electron voltammetry (monitoring the transfer of electrons to/from the probe) and PPBPs bind their targets reversibly, the result will be a biosensor suited to continuous analyte monitoring. After optimization of the sensor in vitro, we will validate the sensors in a complex cellular environment by detecting the flux of glycine and other neurotransmitters in live brain slices. Altogether, these biosensors will be an important step towards the holy grail of biosensing; sensors capable of on-demand, continuous, and non-invasive sensing of any analyte of choice.

Long-lived plasma cells (LLPCs) produce durable antibodies against a wide variety of pathogens, and are associated with long-lasting immune protection. LLPCs arise from T cell-dependent structures in secondary lymphoid organs known as germinal centers, whereby naïve B lymphocytes undergo rapid proliferation and mutate their Immunoglobulin genes. Cells that successfully generate high-affinity antibodies are selected to live, becoming either memory B cells or plasma cells (PCs), which in turn give rise to two main populations: short-lived plasma cells (SLPCs) and LLPCs. SLPCs are found in secondary lymphoid organs, bone marrow and mucosal tissues, where they eventually become LLPCs. Despite the importance of this cell type for protective responses throughout life, the factors driving each PC population is not fully understood. Moreover, a still unaddressed question in the field is why only a few clones of SLPCs convert into LLPCs, and the mechanisms driving this differentiation. Thus, this project aims to define the rules governing SLPC-LLPC transition. Through the combination of transgenic PC-reporter mice, cutting-edge techniques and tools available in Victora’s Lab, we will be able to specifically track SLPCs and LLPCs, providing important clues about their specific niches, clonal composition and patterns of gene expression. Taken together, the innovative approaches presented in the current proposal will faithfully address unsolved mysteries about the biology and dynamics of plasma cells, generating knowledge that will help to better understand the mechanisms driving tissue homeostasis, effective humoral responses and the development of better vaccine-based strategies.

The largest animals of the past and present are suspension feeders, surviving by filtering and eating huge quantities of some of the smallest food in the ocean. This strategy has evolved repeatedly in a wide diversity of animals—from sponges to mammals—and is thought to be the most efficient feeding option for supporting the body mass of ocean giants like whales and the largest sharks. Despite the charisma and protected status of many large-bodied suspension feeders, we know surprisingly little about their ecologies and the tools they use to process water on industrial scales. This is largely due to challenges of studying these animals and their filters in the wild. We integrate a variety of disciplines and scales in the study of suspension feeding in the basking shark, a cosmopolitan species and the second largest fish, reaping >30 kg of plankton per day. We will use animal-borne tag sensors, drones and swarms of underwater robots to capture and dissect basking shark feeding events, to quantify the mechanics, behavior, and hydrodynamics of foraging. These data will be integrated with morphological and anatomical investigations of basking sharks’ fundamental filtering tools—stiff bristles called gill rakers, lining the throat by the tens of thousands—using advanced bioimaging and engineering techniques to quantify their geometries and materials. Behavioral and material data will converge in multi-scale optimization models of flow and filtration, in digital simulations and performance tests of 3D printed biomimics, building from fine-scale biological architectures to explore hypothetical shapes and arrangements, using machine learning to integrate model predictions from micron to meter scales. Seeing a high-performance biological system through the lenses of materials science, animal behavior, robotics and architecture, we gain a unique mechanistic window into the interaction of basking sharks with their environment, and the tools and energetics of a specialized ecology that has existed for millions of years. Exploiting this intersection of disciplines, we will learn how mineralized materials can be modified into high-throughput filters; create new integrated approaches and tools to characterize complex bio-architectures and monitor large aquatic animals in their habitats; and gain bioinspiration for new filter design in a wide range of applications.

Fertilization is a dynamic process that involves the approach, binding and fusion of haploid gametes, the sperm and the egg. To date, only three proteins have been shown to be essential for fertilization in vertebrates: Izumo1, Juno, and CD9. In mammals, the binding of Izumo1 and Juno mediates sperm and egg interaction, yet many key regulators of fertilization have likely been missed, possibly due to redundant functions, since this process involves the coordination of multiple proteins. Therefore, it is instrumental to apply new tools to uncover new molecular regulators of fertilization. I propose to use a multifaceted approach that includes subcellular proteomics, quantitative in vivo imaging and genetics to gain a comprehensive understanding of the dynamics and molecular mechanisms regulating fertilization. Such approaches are technically challenging in the mammalian system, where fertilization occurs internally and access to a large number of eggs is limited. The zebrafish model provides many advantages for fertilization studies, since fertilization occurs externally, and many eggs and sperm can be collected from adults for proteomic studies. High resolution imaging studies are facilitated in the zebrafish (Danio rerio) system due to the presence of the micropyle, the single sperm entry site in the zebrafish egg. These features result in the ability to address long-standing questions in the field with emergent methodologies that will provide a new mechanistic and biophysical understanding of gamete binding and fusion.

During an infection, antibodies undergo drastic improvements in their binding-affinity with the antigens. These changes are driven by affinity maturation, a process taking place in the germinal centers (micro-structures found in lymphoid organs). In these centers, the antibody-producing B-cells pass through multiple cycles of somatic hypermutation (division and mutation), and clonal selection (competition for antigen availability). This process is similar to Darwinian evolution and occurs in parallel with the actual evolution of the pathogens. While high-throughput sequencing has made the study of antibodies in-vivo much easier, the lymphoid organs, where affinity maturation takes place, do not lend themselves to longitudinal studies. In-vitro methods bypass these limitations, but they are often focused on the final product and discard the less efficient branches. This project aims to better understand affinity maturation with the help of an in-vitro experiment. The experiment, based on yeast display methods and FACS sorting, can measure the affinity of antibody's fragments in a high-throughput way. Coupled with barcoding, this allows us to study and evolve, in parallel, multiple branches of the evolutionary tree. The first part of the project consists in measuring the affinity of the clones in a given B-cell lineage, and estimate to which extent the affinity landscape associated with a specific antigen allows to predict the form of the immune response. This will be applied to studying how the evolution of the antigen impacts the development of antibodies.

The state of brain circuits is changed by neuromodulatory signals such as dopamine, serotonin or acetylcholine, which are released in spatially and temporally precise, phasic or tonic patterns depending on the physiological context. These neuromodulators act on multiple receptor classes, exerting diverse physiological effects through distinct signaling pathways. Obtaining a clearer picture of the function of neuromodulatory-driven signals in neural information processing and plasticity requires methods for remote-control of specific neurotransmitter receptors with high temporal precision in defined neurons and in behaving animals. We combine labs with expertise in synthetic chemistry and photochemistry (Ellis-Davies) and behavioral neuropharmacology (Mourot) in order to develop technologies to gain optical control over DA neuromodulation in the freely-moving mouse, for studies of neural circuits and behaviors associated with same-sex social interactions. Synthetic photochemical tools (caged and photoswitchable compounds) are, in principle, able to mimic the timing, amplitude and spread of naturally occurring neuronal signals. However, their in vivo use has been highly restricted, notably because these probes are stimulated by UV or visible light that penetrates tissue very poorly. To overcome this issue, we propose to use lanthanide-doped upconverting nanoparticles (UCNPs), which will be functionalized with caged/photoswitchable ligands for glutamate and dopamine receptors, enabling non-invasive control of the very receptors of the brain in behaving mice. We will further combine near-infrared photocontrol with chemo-genetic strategies to achieve optical control of receptors in defined neuronal targets. Leveraging UCNPs in this way will provide a platform for the interrogation of dopamine-related neuropsychiatric disorders in groups of animals in naturalistic environments, and will represent a milestone in our understanding of how dopamine impacts a variety of personality traits and same-sex interactions. The strategies developed here will be applicable to most receptors, neural circuits and animal models, thus our project bears enormous potential in changing the landscape of our understanding of neuromodulation, both in health and disease.

Understanding how vision guides behavior is a key goal of neural systems research. This project will use long-term behavioral assays and electrophysiology to investigate the perceptual rules and neural dynamics underlying camouflage behavior in the cuttlefish – an animal which uses vision and neural control of skin patterning to ‘match’ itself to the background visual scene. As the cuttlefish skin provides a readout of the animal’s perceptual state, this system presents an unparalleled opportunity to study visual processing and complex motor control in the behaving animal. Using large-scale high-resolution imaging techniques to record and reveal the dynamics of skin ‘pixels’ across the body, I aim to determine the behavioral rules and visuomotor computations that govern camouflage behavior. First, I will map the transformation of visual input into skin pattern output by systematically varying the visual scene of the animal’s home environment during long-term behavioral recordings. This will reveal which statistical properties of visual stimuli are reproduced in skin patterns, at single chromatophore resolution, and how chromatophore motor units are dynamically orchestrated to assume these patterns. Next, I will investigate the neural mechanisms of pattern generation using ex vivo electrophysiology in the pathway purported to control this behavior. I am convinced that the study of visually-guided body patterning behavior will yield fundamental principles of visual perception and complex sensorimotor processes in general.

Dendrites govern feature selectivity in neurons by integrating and subsequently filtering thousands of synaptic inputs to decide overall spike output. A classic example of this relationship is observed in place cells (PCs) in the hippocampus, which spikes when an animal enters a specific position in space. Recent evidence based on two-photon imaging suggests place-related activity is driven by dendritic plateau potentials, however, the precise temporal relationship between such dendritic electrical activity and somatic output in vivo is still unknown. Moreover, coding in the hippocampus, occurs at the population level – sequences of PCs encode trajectories, and are replayed during consolidation. Replay occurs during transient high frequency LFP oscillations call sharp wave ripple (SWR). How is dendritic activity coordinated across SWR-sequences? What is the temporal structure of such signals? These questions remain unanswered because the small size of dendrites have precluded conventional electrophysiology to date. Here, we aim to map CA1 basal dendritic voltage dynamics in head-fixed and freely behaving animals using high-density transparent NanoElectrode Arrays (NEA) comprising of vertical metal-coated silicon NanoNeedles (dia ~100 nm; height ~60 um) and interleaved planar electrodes interfaced to custom CMOS amplifiers, and integrated onto a transparent glass cannula. This new device will enable optical, and, high-density intracellular and extracellular electrical recordings of dendritic dynamics during place field formation. We will additionally correlate intracellular (NanoNeedles), local-field-potential (LFP) (planar electrodes), and two-photon Ca2+ imaging through the transparent cannula to decode how basal dendritic activity shape PC sequences in head-fixed mice running on a circular track. Finally, once we have ascertained the NEA dendritic waveforms in head-fixed mice, we will map dendritic electrical activity in freely-moving animals performing a working memory task flanked by sleep sessions (to enable SWR recording). Using this novel NEA we will ascertain how the LFP and intracellular basal dendritic activity interact to encode place fields during memory formation and replay.

Voltage-gated sodium channels (VGSC, Nav) govern membrane excitability by initiating and propagating action potentials in nerve and muscle tissues. Mutations in Nav channels are directly linked to numerous human diseases, including epilepsy, arrhythmia, and pain. Understanding the gating mechanism of Nav channels is critical for both basic research and pharmaceutical applications. In recent years, several structures of Nav channels that were purified in detergent micelles were revealed by cryogenic electron microscopy (cryoEM). However, the structure of the resting state, which requires the presence of the transmembrane electric field, has been unattainable due to the technical challenges of maintaining the membrane potential during sample preparation. Here we propose an innovative way to prepare cryo-samples of Nav channels on in situ neuronal membranes and further elucidate their resting states by sub-tomogram averaging methods. Novel systems for sample preparation and data collection will be developed to obtain high-resolution structural information on Nav channels in distinct functional states in situ. The strategy can also be generalized to investigate other membrane proteins in a native membrane environment. Achieving the proposed aims will provide unprecedented insight into the Nav channel gating function, and provide useful tools for studying the structural biology of membrane proteins in general.

The malaria disease represents a significant global burden. Despite major advances in its treatment and control, rising resistance to front-line therapies makes the demand for innovative solutions more important than ever. To identify possible intervention points, we need a firm grasp on the processes that govern the parasites complex lifecycle. Central to the evolutionary success of the malaria parasite is the rapid invasion of erythrocytes and subsequent replication. During invasion, the parasite utilises intricate machinery coordinated by a sequence of receptor-ligand interactions, allowing the invagination of the erythrocyte membrane. Inside the erythrocyte, transient structures essential for invasion are rapidly disassembled. These include a double membrane structure termed the inner membrane complex. Despite their importance, our understanding of these transient structures is limited. I propose a correlative approach to understand receptor-ligand mediated erythrocyte invasion and subsequent disassembly of the inner membrane complex. I aim to directly visualise and analyse these processes in situ. This will be achieved using super-resolution fluorescence microscopy to guide specimen FIB-milling for electron cryo-tomography and subtomogram averaging. Simultaneously, studying the inner membrane complex over time in combination with mass spectrometry will allow me to identify and target key players in the disassembly process. Finally, using cryo-tomograms, I will generate a series of atlases of protein and organelle positions within the parasite and host cell. These will provide details of the alterations in the molecular landscape during and post invasion.

Brown adipose tissue (BAT) thermogenesis is essential for survival in cold temperatures. To meet the energetic demands of this process, BAT relies on efficient energy supply from the circulation as well as from intracellular triglyceride stores in lipid droplets. The fatty acids stored in triglycerides can be liberated by lipolysis and serve as fuel for mitochondrial thermogenesis. However, high intracellular levels of free fatty acids are detrimental for cellular survival, demanding for efficient transfer processes. Microscopical analyses indicate a tight interaction between mitochondria and lipid droplets, yet even the fundamental triggers leading to the initiation of contact site formation as well as their physiological role remain elusive. In this proposal, I therefore want to examine the hypothesis that molecular complexes mediating interactions between mitochondria and lipid droplets are formed in activated BAT, and that these contact sites are critical for BAT function. To achieve these goals, microscopical interaction studies will be employed to shed light on the conditions triggering contact site formation. The molecular machinery mediating the interaction will be identified using an APEX-proximity labelling approach. Genetic manipulation will be employed to examine the role of the proteins identified in this screen for initiation and maintenance of contact sites. The relevance of these proteins for BAT function will then be analyzed using CRISPR-Cas9 technology in primary adipocytes and mice. Conclusively, the proposed approaches will not only help understanding the mechanisms involved in organelle contact site formation but also define their physiological role.

From invertebrates to mammals, social behavior plays a fundamental role in the survival of species throughout the animal kingdom. In order to take advantage of past interactions with conspecifics, the brain has to encode, consolidate and recall social memories. Recent findings suggest that the hippocampal region CA2 is essential for social memory, but the nature and the stability of information stored in this circuit remains enigmatic. I will utilize the Egyptian fruit bat, a highly social mammal which develops long-term social networks within the colony, to ask how the long-term and dynamic representation of social information is encoded in CA2. I will adopt advanced behavioral measurements combined with machine learning methods to monitor and characterize social interactions and their associated vocalizations within a natural colony of bats over long periods. To investigate the existence and properties of social coding in CA2, I will take advantage of chronic wireless calcium-imaging in freely behaving and flying bats during behaviorally relevant social interactions as well as during sleep. By combining an ethological approach with cutting edge technologies for detailed measurements of long-term behavioral and neural dynamics, I will aim to uncover fundamental properties of social cognition in the mammalian brain.

Functional enzymes exist in an ensemble of conformations. Protein dynamics, including the transition between different conformational states, have been shown to play an important role in enzymatic functions. Additionally, conformational ensembles are thought to play a key role in enzyme evolution. For example, the evolution of a new catalytic function may depend on a shift in the conformational ensemble. This theoretical model is widely acknowledged, however, there is currently little experimental evidence linking conformational and catalytic ensembles with evolution. Thus, developing dynamic, high-resolution and single-molecule level views of enzymes is a pivotal step to advance our fundamental understanding of enzyme evolution. In this proposal, we aim to capture a detailed resolution picture of conformational and catalytic changes across the evolutionary transitions between divergent enzyme functions. This will be achieved via the integration of diverse cutting-edge approaches, including: directed evolution (DE), single-molecule enzyme kinetics, dynamic structural biology, and computational simulations. We will characterize multiple series of enzyme intermediates that constitute complete adaptive transitions from one function to another. Conformational ensembles and motions will be characterized using serial femtosecond crystallography (SFX) combined with extensive molecular dynamic (MD) simulation. Catalytic fluctuations and heterogeneity will be experimentally measured using single-molecule enzymology techniques, and conformational changes in the reaction will be determined by kinetic-crystallography. Furthermore, we will identify the intramolecular amino acid networks that are associated with changes in conformational motions and ensembles. Finally, all experimental data will be integrated into an integrated computational model in order to describe the process of enzyme evolution as topology change in the conformational energy landscapes. This work will lead to an unprecedented experimental and computational understanding of the interplay between conformational and catalytic ensembles of enzymes and their evolutionary dynamics. These in-depth molecular views of enzyme evolution will not only advance our basic scientific knowledge but also lead to technological advances for the design and generation novel enzymes in the laboratory.

Animal-bacterial interactions are widespread and regulate diverse vital processes, including nutrient uptake, immune system development and tissue morphogenesis. Although crucial for animal biology, little is known about the ancestry of animal-bacterial interactions or their influence on animal origins and evolution. As the closest living relatives of animals, choanoflagellates provide key insights for reconstructing animal origins. In the model choanoflagellate S. rosetta, molecules secreted by bacteria induce two life history events – multicellular development and mating (Alegado et al. 2012, Woznica et al. 2018). However, unlike in animals in which bacterial symbionts are stably enclosed within epithelial structures, the S. rosetta life cycle responds to diffusible cues from environmental bacteria. A new choanoflagellate species, S. monosierra, discovered by the host lab forms large spherical colonies containing a stable microbiome (Hake et al., in prep). Reducing the bacterial load reduces S. monosierra colony size without reducing cell number, suggesting the microbiome regulates colony morphogenesis. This proposal seeks to harness this new bacteria-containing choanoflagellate to investigate the ancestry of animal-bacterial symbiosis and its influence on epithelial morphogenesis. First, I will define the precise symbiotic bacteria that regulate S. monosierra colony size. Second, I will investigate the molecular cues and cellular mechanisms that underlie bacterial-dependent control of S. monosierra colony size. Last, I will explore choanoflagellate diversity to assess whether a stable microbiome was present in the last common ancestor of choanoflagellates and animals.