A major societal challenge in the Western world is the dramatic increase in chronic inflammatory disorders manifesting at barrier sites, including allergies, asthma (lung), psoriasis (skin), and inflammatory bowel disease (IBD; intestine). Recent advances identified a new group of innate immune cells primarily residing at such barrier surfaces, which were named innate lymphoid cells (ILC). The major task of ILC is to guard, repair and protect the tissue barrier. However, chronic activation of ILC can promote inflammation and contribute to inflammatory diseases. ILC like other immune cells have the ability to change their function and convert into different cells of the same type. This ability is called plasticity. The underlying reasons for such functional conversions are not known, but ILC converting into other cell types are often involved in the development of inflammatory diseases. Here we propose that ILC plasticity may have evolved to maintain immune functions in nutrient low conditions as experienced in starvation to allow for a thriftier immune response. However in the context of high nutrient availability experienced in the western world, the capacity to convert to different cell types may have rendered plastic ILC function pathogenic resulting in chronic inflammation. To address our hypothesis we will investigate whether conversion of ILC is dependent on the nutrient status and if having more nutrients available will result in detrimental ILC function e.g. more allergies. Thus with our research we aim to contribute to the understanding of the dramatic increase in chronic inflammatory disorders and to identify novel treatments for such diseases.

Enhancement of the immune system by immunotherapies is highly effective in inducing durable responses to cancer. Colorectal cancer (CRC) is the third most common cancer in both men and women and accounts for nearly 10% of all cancer-related deaths. This points to the urgent need for the development of therapies that are effective in patients suffering from this disease. Tim-3 and CEACAM-1 are two inhibitory molecules that are highly expressed in the gut, where they regulate development of the gut immune system. However, due to the complexity of the specific microenvironment of the colon, little is known regarding the specific interactions between colon cancer cells, the myriad of immune cells within the colon, microbiome and food metabolites that may be regulating immune response to CRC. By using mouse models for CRC, I aim to elucidate these interactions by:(1) comparison of immune response to tumor formation in the orthotopic location (colon) vs. ectopic location (flank);(2) Investigation of the role of the inhibitory molecules TIM-3 and CEACAM-1 on various T cell and myeloid cell subsets, using a wide variety of genetically altered mouse strains, and test the efficacy of antibody blockade of TIM-3 and CEACAM-1 in the various models in order to realize what might be the correct strategy for optimal clinical benefit; (3)analysis of the regulatory gene network downstream of TIM-3/CEACAM-1 function. By understanding the immune response against CRC in its physiological context, and by utilizing state-of-the-art facilities, in vivo and ex vivo techniques, I intend to uncover new avenues for therapeutics against CRC, a cancer that hitherto has been resistant to immunotherapy.

Multicellular organisms use asymmetric cell division (ACD) to generate diverse cell types during development. In contrast to the mechanism revealed in animals, our knowledge on plant ACD is limited. Current models focus on the plant-specific microtubular structure, the preprophase band (PPB), in regulating oriented division. However, recent studies suggest a more fundamental role of proteins at the cortical division zone (CDZ), which assemble regardless of the presence or absence of PPBs, during ACD. However, it is unclear how CDZ is formed at a specific cortical domain and how it regulates oriented cytokinesis, which involves rotation or “guidance” of the mitotic apparatus (called phragmoplast). I aim to reveal the molecular mechanism underlying plant ACD, focusing on the assembly and function of the CDZ. Specifically I will: 1. Elucidate the phragmoplast guidance mechanism through advanced imaging and revealing the function of a key kinesin motor at the CDZ. 2. Identify new factors that affect CDZ assembly and phragmoplast guidance. I will use the moss Physcomitrella patens as the model system, which has a short life cycle, simple morphological structures, and efficient genome-editing tools. Both PPB-dependent and -independent asymmetric division take place in moss, which enables the identification of core effectors involved. By combining advanced genetics and high-resolution imaging tools, I will investigate the roles of CDZ in ACD through identifying novel factors upstream of CDZ and dissecting the interplay between CDZ and phragmoplast-involved cytokinesis. In the end, I envision mechanistic comparison with better-studied animal and yeast systems.

General anesthesia (GA) reversibly induces the loss of consciousness. Sleep, especially non-rapid eye movement sleep (NREM), is a physiological unconscious state. Human functional imaging studies have shown parallels (but also differences) between anaesthetized and sleeping brains. These observations raise the possibility that anesthetics exert their effects through activating the sleep circuitry. However, to date, it remains unknown which neurons or circuit in the complicated sleep pathways are bona fide anesthetic targets. Here, we hypothesize that GA activates specific neuronal ensembles in the brain, and activities of anesthesia-activated neurons (AANs) are sufficient to induce unconsciousness. Our lab developed an innovative technology called CANE (capturing activated neuronal ensembles) that enables me to selectivity and efficiently capture activated neurons and express desired genes in these neurons. I have successfully captured AANs located in the extended supra-optic area in ventral hypothalamus using CANE. Here, I will examine the intrinsic electrophysiological properties as well as anesthetics-evoked responses of CANE-labeled AANs using slice electrophysiology. I will perform in vivo recording of AANs during natural awake/sleep cycles and upon exposure to anesthetics using photo-tagged optetrode recording. Furthermore, I will map the input and output circuits of AANs using CANE-based tracing technology, and determine the causal functions of AANs on brain state transitions using chemogenetic/optogenetic activation or inhibition approaches. I expect to gain ground-breaking new insights into the neural mechanisms underlying anesthesia.

Chloroplasts (plastids) and mitochondria, which are derived from endosymbiosed bacterial ancestors, proliferate by binary division of pre-existing organelles. This division is carried out by separate ring-shaped macroprotein complexes known as the plastid- and mitochondrial-division machineries. Although many studies have examined plastid and mitochondrial division, much about the components and molecular mechanisms of the division machinery of each organelle remains to be discovered. To address these questions and to drive the field into a new stage, I will carry out the following research projects: (1) Molecular characterization of the components of the plastid- and mitochondrial-division machinery; (2) Investigation of the kinetic mechanisms of the plastid- and mitochondrial-division machinery at single-molecule resolution; (3) Elucidation of the molecular mechanism of PD/MD ring filament synthesis by glycosyltransferases PDR1 and MDR1; (4) Comprehensive characterization of all components involved in organelle proliferation mechanisms, not only for plastids and mitochondria but also for peroxisomes, Golgi, vacuoles, and the ER, via genome- and system-wide analyses. These proposed studies will provide insight into how the division machinery drives the constriction of division sites in these organelles, at single-molecule resolution, and how free-living cyanobacteria and alpha-proteobacteria evolved into plastids and mitochondria. Organelles are specialized subunits found in eukaryotic cells; thus, comprehensively investigating organelle proliferation mechanisms will reveal fundamental principles about organisms ranging from primitive eukaryotes and plants to animals.

Past research has identified epigenetic mechanisms including histone modifications as key regulators of transcription and implicated them in cellular differentiation. But while our knowledge about their cell type specific distribution and its influence on transcription has constantly increased, we still know very little about the orchestration of epigenetic and transcriptional changes as they occur during differentiation. To gain a global understanding of the order of events, we aim to identifying changes in gene expression and main transcriptional repressive and promoting histone modifications in cells undergoing differentiation. For this we will develop a novel approach to examine the distribution of histone modifications on a single cell level adapting a method based on antibody targeted micrococcal nuclease (ChIC-seq), which will lead to a modification dependent enzymatic digestion of the DNA. In comparison with existing single cell ChIP approaches this method lacks a precipitation step, leading to minimal material loss per cell, while allowing the use of the variety of histone mark specific monoclonal antibodies. Adapting this approach to the previously described method for co-acquisition of transcriptomic and epigenomic information from a single cell existing in the lab, will allow the normalization of the histone state on the transcription status of the cell. To analyze the order of changes associated with cell differentiation, we will use single cell RNA seq analysis pipelines (RaceID + StemID) to identify and enrich for cells in between two specific differentiation stages. look specifically at cells undergoing differentiation.

Pancreatic ductal adenocarcinoma (PDAC) depends heavily on nutrient-scavenging pathways such as macropinocytosis and autophagy to obtain the necessary nutrients for biomass accumulation and enhanced cell proliferation. These pathways converge at the lysosomal level were macromolecules get broken down into simple metabolites that can be reutilized. As a result, lysosomes can act as a cellular depot controlling the availability of nutrients, impacting cell metabolism. Despite the biological relevance of this mechanism, pancreatic cancer-associated lysosomes remain poorly characterized. My main aim is to identify the structural and functional components of pancreatic cancer lysosomes in vivo. For that, we will generate an in vivo mouse model of pancreatic cancer bearing a lysosomal tag that allows for their rapid and efficient isolation. Using this model, we will characterize structurally and functionally the lysosomes from different stages of tumor development. By using an in vivo CRISPR/cas9 screening, we will identify lysosomal proteins essential for lysosome formation, metabolic availability and cell proliferation, potentially revealing pancreatic cancer specific vulnerabilities. Finally, we will perform quantitative metabolomics and metabolite-tracing experiments to define specific metabolites recovered through the lysosomal pathways and determine their fate within the metabolic network, shedding some light in the uncharacterized process of cancer metabolic rewiring. This project will help us understand how lysosomes orchestrate the metabolic adaptation of pancreatic cancer, which may uncover new targets and lead to the development of new anticancer strategies.

Breast cancer frequently metastasizes to bone where it leads to osteolytic bone degradation and poor clinical prognosis. Nevertheless, therapeutic options to interfere with this process are scarce as the underlying mechanisms remain unclear. Most current research focuses on the cellular and molecular signaling underlying bone metastasis, but breast cancer cell interactions with the bone mineral matrix, a composite of collagen fibers reinforced with nanoscale crystals of hydroxyapatite (HA), may be similarly important. However, in the field, there is an ubiquitous lack of (i) high resolution techniques to characterize the multiscale structure of bones as a function of breast cancer progression, (ii) microscopy techniques to identify the underlying changes in bone mineralization pathways in fully hydrated samples, and (iii) model systems to evaluate the functional consequences of varied bone mineral properties on cell behavior largely prevent such studies. Here, we will address these shortcomings and capitalize on the interdisciplinary background of the proposed team in cancer biology and tissue engineering (Fischbach), high resolution structural analysis of hierarchical materials (Fratzl), bone biology and biomineralization pathways (Addadi), and bioinspired materials synthesis (Estroff) to test the hypothesis that breast cancer modifies the hierarchical structure of bones by altering bone mineralization pathways and that the resulting changes in HA nanoparticle characteristics are critical to the pathogenesis of breast cancer bone metastasis. To address this hypothesis, we will pursue three specific aims: Aim 1 will analyze the multiscale structure of bones in mouse models mimicking pre- and post-metastatic stages of breast cancer. Aim 2 will determine the corresponding differences in bone mineralization pathways and Aim 3 will synthesize biofunctional bone-mimetic materials to study the effect of bone mineral matrix properties on tumor and bone cell behavior. This work will broadly impact our understanding of breast cancer bone metastasis and challenge the conventional paradigm of bone metastasis of this disease as solely initiated by cellular and molecular mechanisms. Insights gained from our studies may yield novel therapeutic targets to treat or possibly even prevent bone metastasis in the future.

Animals must constantly integrate information from the environment with prior experience to maximize reward and avoid danger. Such goal-directed functions require the prefrontal cortex (PFC), a brain structure that is extensively interconnected with sensory regions, limbic structures and motor systems. The PFC circuit is thought to orchestrate fear, anxiety, motivation, attention and other behavioral processes that are crucial for survival in a constantly changing environment, and impaired prefrontal functions have been associated with diverse psychiatric conditions including schizophrenia, major depression, attention and anxiety disorders. The PFC interacts with multiple cortical and sub-cortical brain regions through its long-range output projections, originating from defined populations of excitatory PFC output neurons. These neurons send their axons to a wide range of downstream brain regions, including the amygdala, the hypothalamus, the basal ganglia and other structures known to directly modulate the animal’s behavioral state and its responses to the environment. How do these neurons route information to one downstream target or another? What are the genetic, structural and functional changes through which they facilitate the acquisition of goal-directed behavior? Previous work has explored these questions, revealing unique roles for distinct types of PFC neurons in complex behaviors such as working memory function, attentional modulation, learning and memory. However, these studies typically focused on single populations of PFC neurons, while the state of the network as a whole is known to be dependent on the joint function of all of its components. We will combine advanced single-cell genomic analysis (Amit lab) with optogenetic functional experiments, electrophysiological approaches (Yizhar lab), innovative tissue processing and volumetric fluorescence imaging techniques (Kim lab), to understand the organization and function of PFC output neurons and their learning-related plasticity. Our results will reveal the unique molecular, structural and functional properties of defined prefrontal cortical output pathways, and allow for the first time a system-level understanding of the functional dynamics supporting the regulation of goal-directed behavior by this complex circuit.

How environment shapes cellular responses is an important field in epigenetic research, very much owing to the role of certain epigenetic modifiers as metabolic sensors. Metabolic signaling to chromatin is linked to local concentrations of small metabolites which modulate the function of enzymes using them as a cofactor, some of these have important regulatory roles in defining transcriptional responses. During stem cell activation and lineage commitment, a metabolic switch occurs from anaerobic glycolysis to oxidative metabolism as a source of energy. These transitions are known as metabolic reprogramming, and are paralleled by a major epigenome reorganization which assists the establishment of a specific pattern of gene expression in the differentiated cell. These events are hallmark of cellular differentiation, yet the molecular links between them are poorly understood. Within the CHROMET project, we aim to understand the connections between metabolic and epigenetic reprogramming during stem cell differentiation in single living cells. We propose to develop dedicated optical techniques based on Fluorescence Lifetime Microscopy to track the endogenous redox metabolites NADH and FAD, creating maps of their local binding within the differentiating cells. We will identify subnuclear microenvironments enclosing these metabolites, and functionally characterize them by analyzing their local bindings to key epigenetic remodelers using them as a cofactor. We will also investigate the role of nuclear redox metabolism in the relocation of developmentally regulated genes within the nucleus, which takes place during lineage commitment. CHROMET project is stablished through the collaboration between a multidisciplinary team including Lorena Aguilar Arnal, with strong expertise in molecular and cellular biology focused on epigenetic research, and Chiara Stringari, who is developing new methods for non-invasive imaging of endogenous metabolites in living cells and tissues.

Some four and a half billion years ago, primitive proteins spontaneously phase separated from their environment, like oil from water, to form liquid droplets, which became crucibles for the evolution of the chemical processes that underlie life to this day. This is how the Russian biochemist Alexandr Oparin postulated that life began to evolve, almost a century ago. Very recently, the droplets that Oparin predicted have been discovered and they are composed of molecules resembling the primitive proteins he imagined would have been found in the “primordial soup”. These droplets have been shown to serve a number of important functions and proteins that compose them underlie neurological diseases, including Parkinson’s and Huntington’s diseases and ALS, among others. How many such structures, called non-membrane organelles (or NMO), could exist in the cell and what are their functions? What are the physical rules and chemical information encoded in the sequences of MNO-forming proteins that dictate their unique properties? What happens if NMOs are disrupted? These are the questions we have set out to address in this proposal. The central aim of our proposal is to create a compendium of NMOs in a model of the eukaryotic cell; the bakers yeast. Yeast is an ideal model because it shares many genes with humans, notably those that are most primordial and those that become dysfunctional with age and in diseases such as cancers, and neurodegeneration. We will use massively parallel and automated experimental and computational and theoretical approaches to determine protein sequences that code for their ability to form MNOs of different types and predict the biological function of specific MNOs. We have shown how certain types of cell regulation including gene function and spatial organization of large cell structures depend on NMOs. The compositions of NMOs are surprisingly rich in protein molecules that when mutated, are known to underlie neurodegeneration. We hope to define how these mutant proteins cause dysfunction, perhaps by disrupting the normal functions of NMOs, and what kinds of strategies could re-stabilize them. Finally, the rules of NMO formation and function we elucidate in yeast will allow us to make predictions about similar structures throughout the tree of life, including in humans, ultimately establishing another axis of order that defines life.

Transport and degradation of marine particulate organic carbon (POC) has been widely studied in oceanography, yet most research has focused at the bulk scale. While it is known that microbes are key players in controlling the fate of POC, we lack a mechanistic understanding of the biochemical and physical interactions by which microbes drive POC degradation . In this project I will explore these microscale processes by focusing on the microbial dynamics around particles. To achieve this, I will develop a 'milli-fluidic' experimental system to permit the visualization of single POC aggregates in a closed-loop chamber. I will observe aggregate decomposition under flow as a function of time and treatment, and I will quantitatively characterize microscale bacterial attachment, bacterial growth, biofilm formation and enzyme function, as well as the coupling among these processes. These unique experimental data will inform a new mathematical model of POC degradation kinetics to predict the conditions and depths at which POC is remineralized in the ocean. By unraveling the dynamic microscale interplay among biology, biochemistry, and physics in the degradation of marine particles, this work will bring about a more fundamental, mechanism-based understanding of particle flux and the carbon cycle in the ocean.

Seabirds have the longest migrations on earth and can travel 8 million km in a lifetime, yet how they navigate across a seemingly featureless ocean is still one of the greatest puzzles in nature. Evidence from mammalian and insect systems shows that animals adjust their behavior in response to infrasound and a handful of studies have suggested pigeons may use infrasound for navigation. These low frequency sound waves can propagate over hundreds of kilometers, creating “hills” and “valleys” of an infrasoundscape that birds may use to navigate, like a topological map. When combined with meteorological and oceanographic models, these maps can be modeled to create real time soundscapes that individual seabirds could use in movement decisions over spatial scales. By combining a network of 60 international atmospheric infrasound and hydro-acoustic monitoring stations that detect signals from around the globe with a database of over 15,000 seabird movement tracks, we will have a unique opportunity to explore the role of atmospheric and oceanic infrasound in navigation, respectively for aerial and aquatic species. The mechanisms allowing animals to detect low frequency sound has been identified in other taxa, and our study will examine how seabird sensory organs may capture infrasound. The development of an innovative movement framework grounded in landscape ecology will allow us to assess determinants of large-scale movement, notably the effect of infrasound in directing migration and commuting trips in the open ocean. Furthermore, novel biologging devices, which can detect sound and meteorological parameters, will be used to simultaneously capture movement, infrasound and weather conditions to examine individual movement decisions at fine scale. Finally, interspecific comparisons will assess the relative importance of infrasound for seabird navigation, with respect to phenotypical and phylogenetic differences, thus offering a complete assessment of the physiology, behavior and physics underpinning the use of infrasound in navigation.

Sleep deprivation disrupts memory consolidation, and alters gene transcription in the cortex and hippocampus. We hypothesize that sleep deprivation-induced deficits in memory processes are caused in part by misregulation of the molecular circadian (~24hr) clock. Until now, experimentally testing this hypothesis has been a major challenge. Specifically, the molecular clock controls sleep and wake behavior through effects on the suprachiasmatic nucleus and other sleep-regulatory circuits. Furthermore, sleep and wake cause widespread, simultaneous changes in transcription, translation, neural activity, neuromodulation, and hormone release. Thus ascribing functional causality to one specific state-dependent variable (and in particular, clock gene expression) has proven difficult. Here, we propose to develop and use novel optogenetic tools to rapidly up- or downregulate individual clock genes in vivo, in a circuit-specific manner, independent of the animal’s behavioral state. We will use cutting-edge computational models and bioinformatic analyses to optimize optogenetic regulation of clock genes in the brain. We will test our hypotheses using both traditional and novel optogenetic tools, computational modeling, bioinformatics, behavioral analysis and electrophysiology. This can only be achieved by a synergistic collaborative intercontinental, interdisciplinary research team: a group at the University of Düsseldorf (Germany) will develop optogenetic tools, a group at the Korea Advanced Institute of Science and Technology (South Korea) will carry out mathematical modeling and bioinformatics analyses of CCGs, and groups at the University of Michigan (United States) will study effects on cortical plasticity, and a group at the University of Groningen (Netherlands) will examine effects on hippocampus-mediated memory.

Asthma is a clinically heterogeneous disorder caused by genetic and environmental influences that remain poorly understood. Promoter variations that induce over-expression of the Orosomucoid-like 3 gene (ORMDL3) in humans are the strongest genetic associations with severe childhood asthma. Orm-family proteins are a component of an essential enzyme complex, the SPOTS complex, for Serine Palmitoyl-transferase, Orm, Tsc3, and Sac1 complex, which catalyzes the rate-limiting step for de novo sphingolipid synthesis. Orm-family proteins act as inhibitors of Serine Palmitoyl-Transferase (SPT) and play an integral role in sphingolipid homeostasis. Elevated expression of ORMDL3 inhibits the synthesis of sphingolipids that appear to be protective against asthma and other inflammatory diseases. These observations raise the exciting possibility that sphingolipid synthesis may be a point of intervention that is orthogonal to existing therapies. However, the molecular architectures of the SPOTS complex and the Orm proteins, as well as the details of their functional roles in sphingolipid metabolism, are entirely unknown. I propose to determine the structure of the SPOTS complex in different functional conformations using cryo-electron microscopy (cryo-EM) and to characterize the mechanisms governing sphingolipid homeostasis with biochemical methods. These results will improve our understanding of sphingolipid homeostasis and the pathogenesis of asthma and other inflammatory diseases.

Preservation of genome integrity is fundamental to the maintenance of life. Double-strand breaks (DSBs) are a particularly lethal form of DNA damage and cells have evolved conserved and dedicated pathways to repair the same. Although important for cell viability, recent studies have shown that DSB repair can also be a potent source for mutagenesis. While cells in all domains of life are capable of faithful repair via homologous recombination, eukaryotes and some bacteria also employ an alternative, end joining pathway for DSB repair. This pathway is error-prone, but it avoids the lethality of a DSB, making it important. End joining has been well studied in eukaryotes. However, this pathway was relatively recently discovered in bacteria and its mechanism of regulation remains unclear. The objective of this proposal is to study the regulation of error-prone DSB repair in the opportunistic pathogen, Pseudomonas aeruginosa, which encodes for an active end joining repair pathway, and assess the role of such repair in bacterial growth and survival during stress. Using single-cell and single-molecule fluorescence microscopy in combination with genome-scale assays, this study aims to comprehensively elucidate the mechanism of end joining repair in vivo and understand how cells regulate pathway choice between homologous recombination and end joining-mediated DSB repair. Given that end joining repair is conserved across several pathogenic bacteria as well as eukaryotes, this study should provide novel insight into general principles of mutagenic repair and its likely impact on antibiotic and stress resistance.

In this project, we will provide new insights into the behavioral strategies, biomechanics and motor control animals use to perform complex versatile and adaptive functions and provide novel bio-inspired robot technology for solving complex motor control problems.
Despite the constraints imposed by their miniature brains and bodies, dung beetles are capable of the most impressive
feats of motor control and navigation. Beetles of the species Scarabaeus galenus are able to move large objects – dung balls that can exceed 10 times their own weight – accurately between a food source and their burrow by combining celestial compass orientation with step-counting. In addition, they are able to use their specialized body and legs to dig burrows, manipulate different dung types and move them over different types of terrain (e.g. sand or grass) using a variety of locomotor patterns. How do they achieve these complex versatile and adaptive behaviors when moving over difficult terrain? Our project aims to address this through our integrative approach which combines behavior (EB), biophysical, kinematic and biomechanical analyses (SG) of S. galenus, and computational motor control and robotic modeling (biorobotics, PM).
Research on behavior and neuro-biomechanical functions of insects has, until now, largely dealt with few behavioral modes – like walking, climbing, or navigating – in isolation. We will launch a major research effort aimed at uncovering behavioral strategies, biomechanics, and computational motor control that enable the miniature brains and bodies of insects to effectively generate multiple adaptive behavioral modes. Our behavioral experiments, both in the field and in the lab, will investigate locomotion, navigation, food transportation, and their combination in S. galenus. The data from these experiments will lay the foundation for novel biophysical, kinematic and biomechanical analyses, and computational motor control development. Simultaneously, biomechanical principles underlying the different behavioral modes will be used to shape the behavioral experiments and guide the structural design of robotic modeling. We will develop a biorobotic model that will enable us to test our hypotheses about the relationship between structural and mechanical features and how they interact with motor patterns and neural mechanisms to form complex behaviors.

The ribosome is the universal translation machine, which makes all the proteins in the cell, including its own. Much is known about ribosome structure and assembly pathway. However, cell-free synthesis and assembly of ribosomal proteins and rRNA into functional ribosomes has not been reconstituted, which would be essential for establishing a protein-based self-replicating model system of artificial cells, and provide a new means to develop antibiotics. We will address the challenge of cell-free ribosome biogenesis by combining the reconstitution of E. coli translation from purified components developed by Shimizu and Ueda (PURE system), with the miniaturized on-chip DNA compartments introduced by Bar-Ziv's lab. We address following three questions: i) What are the minimal requirements for cell-free ribosome biogenesis?; ii) How is ribosome biogenesis affected by excluded volume interactions, segregation, and macromolecular gradients in the DNA compartment?; and iii) Can DNA brushes coding for rRNA and ribosomal proteins mimic spatial ‘operons’ and promote efficient assembly coupled to synthesis? For these questions, we will attempt to 1) develop a methodology to label and localize cell-free synthesized ribosomal proteins and rRNA; 2) tailor the PURE system to support cell-free ribosome biogenesis reactions; 3) tailor DNA compartments geometrically to direct ribosome biogenesis and measure synthesis and assembly or ribosomal proteins and rRNA using time-lapse fluorescence microscopy; and 4) develop approaches to resolve, physically and biochemically, new from old ribosomes. The combination of molecular biology and biochemistry with soft matter physics and materials science are expected to provide favorable conditions for in vitro ribosome biogenesis and novel means to separate new from existing ribosomes.

A fundamental challenge in biology is to understand how brain cells change in response to experience and how these changes contribute to memory and establishment of other long-lasting behaviors. Changes in neuronal gene expression are important for memory formation, maintenance and retrieval. Recent advances in genome biology have found that three-dimensional (3D) genome organization—genome topology—is critical for transcriptional regulation. We hypothesize that neuronal genome topology provides special “topology codes” for activity-driven transcriptional modulation in neuroplasticity, and abnormal topology codes contribute to cognitive disorders. This project will examine genome topology and transcriptomics in mouse hippocampus and explore how the dynamics of genome topology contribute to the rapid and highly coordinated transcriptional response during learning and the long-lasting changes in gene expression that underlie memory, epilepsy and other enduring forms of neuroplasticity. Our multidisciplinary team with complementary skills in neuroscience, genomics and bioinformatics will take an integrated approach that includes a novel 3D genome mapping strategy for small numbers of cells (Ruan), advanced genetic labeling and biochemical isolation techniques for transcriptional and epigenomic profiling of neuronal ensembles (Barco), and state-of-the-art, multimodal microscopy for nuclear imaging (Wilczynkski). We will develop and optimize our platform in cultured neurons subjected to different stimuli (Aim 1), and we will examine the 3D epigenomic dynamics of the nuclear response to synaptic activity in two well-established in vivo paradigms: epileptic seizure (Aim 2) and the formation of associative memory (Aim 3). The large volumes of high-quality and multiplex genomic data representing dynamic changes in neurons in response to activation will be integrated by comprehensive computational analysis (Aim 4). Predicted genetic loci that have important roles in chromatin topology and transcriptional regulation will be validated structurally by super-resolution nuclear imaging and functionally by genome editing (Aim 5). The results of this effort will open new chapters in learning and memory and epilepsy research by laying a foundation for understanding the dynamic and topological mechanisms of genome regulation in neuronal plasticity in health and disease.

When hearing a sound, structures of the cochlea vibrate. These mechanical vibrations are transformed by sensory inner hair cells (IHC) into electrical activity that travels to the brain where it is decoded. Sounds of different frequencies resonate at different locations along the cochlea. The resonant properties of the cochlea are enhanced by the activity of outer hair cells (OHC) that use metabolic energy to amplify the faintest sounds and sharpen the frequency selectivity. Because of a coupling between cochlear mechanics and neuronal processing, it is still unclear how a complex sound containing multiple frequencies is integrated by the brain and what is the role of the cochlear amplifier.

Here, we will deconstruct cochlear vibrations using optogenetics. We will express channelrhodopsin in IHC and stimulate them with single cell resolution to isolate the neuronal processing. The electrical activity of neurons will be monitored at key locations along the auditory pathway. First, we will evoke auditory response in the brain with optical stimulation of IHC and reproduce the response to a single tone using the appropriate pattern of light stimulation. Second, we will decipher the role of spatial and temporal information in auditory processing. Third, we will use vocalizations and understand how cochlear mechanics shapes the integration of complex sounds at the periphery of the auditory system.

This proposal will provide important information about the role of both cochlear mechanics and neuronal processing in shaping the sensation of sounds. These results will be of particular importance in the development of a new generation of cochlear implants based on optogenetics.