The goal of this proposal is to understand how heterochromatin functions impose major roadblocks to cell fate changes in development and iPS cell reprogramming. Reprogramming somatic cells into pluripotency by the Yamanaka factors has enormous potential in the regenerative field, although the efficiency remains low. Heterochromatin functions as a major epigenetic barrier to cell fate changes and the Zaret lab showed that its erasure on pluripotent genes at the late stage of reprogramming is a rate-limiting step. However, so far little is known about how heterochromatin domains are established, maintained and reset during development and reprogramming. To fill in this gap, we propose to map the dynamics of heterochromatin formation during mouse peri-implantation, when heterochromatin domains are first established, and to map heterochromatin dynamic resetting in reprogramming intermediates. Taking both natural and reprogramming contexts together, we will construct lineage relationships of heterochromatin dynamics at pluripotency genes as they related to transcription. Finally, to gain temporal and mechanistic insights into heterochromatin regulations of pluripotent genes, we will use a live imaging system to assess directly the consequences of systematic knockdown heterochromatin-associated proteins on the transitions between chromatin topologies and transcriptional activation of pluripotent genes and reprogramming efficiency. Our novel approach of combining datasets and analysis of early development and late reprogramming will contribute to designing robust reprogramming strategies with higher efficiencies and realizing full the potential of iPS cells.

The OXPHOS system is the only process in animal cells with components encoded by two genomes, maternally transmitted mitochondrial DNA (mtDNA) and biparentally transmitted nuclear DNA (nDNA). The protein products of both genomes have to physically assemble with their counterparts to build functional respiratory complexes. Therefore, variability in the OXPHOS encoded genes is limited by a physical match constraint. This imposes a close-fitting co-evolution of both genomes challenged by the very different mechanism to generate variability for nDNA (by sexual reproduction, mutation and co-existence of two alleles) and mtDNA encoded OXPHOS genes (by mutation, polyploidy and segregation). Since the simultaneous co-existence of alternative mtDNA encoded alleles for OXPHOS proteins has been shown to be detrimental for the organism, we postulate that the co-expression of nuclear encoded alternative alleles may have similar adverse consequences, and that specific regulatory mechanism prevent them. Indeed, random mono-allelic expression is not rare and was suggested for about 30 OXPHOS genes in mice. We postulate that is part of a sophisticated and multi-level system of quality control and functional testing to select for the best combination for providing metabolic plasticity.
Potential regulatory mechanisms are: selective transcription of one allele per cell or selection of the expressed alleles at the import or assembly of the OXPHOS complexes. For testing this hypothesis, we integrate different skills for the analysis of mitochondrial functional and genetics profiling (Enriquez), single cell transcriptomic (Eberwine) and functional and dynamic analysis (Busch) in mouse (Enriquez) and zebrafish (Mercader). If confirmed, we will set the ground for a novel theory of genetic interaction for OXPHOS function. If discarded, the existence of mtDNA and its particular way of inheritance would be necessary to group those genes for which allelic variability is detrimental.

One of the most basic aspects of sleep is that it happens at a particular time of day. Neuroscientists have known for almost half a century that this consolidation requires the suprachiasmatic nuclei of the hypothalamus (SCN); beyond this point, the circuit remains untraced. Equally mysterious, healthy young humans sleep in a single consolidated bout, while infants and older individuals, as well as laboratory mice, can have highly fragmented sleep. Informed by a combined cellular and circuit-based model for slow-wave or “deep” sleep (Forger Group), we propose to trace the signals leading from SCN to cortical slow waves at both physical and molecular levels, and then manipulate them to artificially consolidate sleep. Starting from cortex, aided by a novel “triple-CRISPR” approach to generate knockout mice efficiently in a single generation, we shall examine roles of individual cortical ion channels and the signaling pathways they regulate in creating these slow waves both in vivo and in newly developed whole-brain culture (Ueda Group). This information will be incorporated into a cellular and then a thalamocortical model of slow-wave sleep (Forger Group). In parallel, synaptic tracing in cleared brain, as well as calcium imaging correlation analysis using miniature skull-mounted microscopes, will establish physical and functional connectivity from SCN to cortex (Brown Group), which can also be tested in whole-brain culture (Ueda group). Modeling these data comprehensively and then optimizing this whole-brain model (Forger group), we can predict and test molecular sources of homeostatic and circadian influence upon sleep, as well as combined transgenic and optogenetic strategies to create consolidated bouts of sleep from the normally fragmented sleep of the mouse (Brown and Ueda groups). In this way, by using the power of large-scale quantitative modeling to explore synergies in sleep-dependent signaling, we hope to provide a starting point for novel multimodal therapies with the capacity to fundamentally alter the sleep-wake landscape.

Enhancers are relatively short DNA sequence elements (<1 kb) that determine the timing, location and levels of gene transcription. They harbor specific binding sites for transcription factors (TFs) that control the enhancer. While the necessity of particular TF binding sites (TFBSs) can be assayed with mutations, it is not yet understood which DNA features in an enhancer sequence collectively give rise to the regulatory activity. To identify the molecular determinants that impart a regulatory activity to a DNA sequence element, we have devised a quantitative experimental paradigm and propose to apply statistical analysis and machine learning approaches to the molecular dissection of a model enhancer.
Specifically, we will use a model enhancer driving patterned spatial expression in the wings of fruit flies (Drosophila). First, we will create tens of variants of this enhancer, introducing mutations along its sequence, and describe their regulatory effect. To this end, we will build an automated imaging pipeline to measure the levels and spatial distribution of reporter gene in the wings. The resulting quantitative expression data in a flat tissue will define a morphospace, i.e., a mathematical multidimensional space representing the possible variation of enhancer activity. Second, we will extract DNA feature sets to quantitatively describe molecular variation along the sequence of our mutant enhancers. This will capture structural changes (DNA shape readout), changes in nucleotide sequence per se (DNA base readout), as well as other features (e.g., TFBSs). Third, leaning on dimensionality reduction techniques and machine learning, we will develop predictive models that describe the relationship between DNA features and morphospace. Finally, we will test our predictions of enhancer functionality using synthetic enhancers in transgenic flies. Because enhancers are complex biological objects, we aim with this proposal at developing appropriate mathematical tools to capture the essence of this complexity.
Since this proposal aims at developing a comprehensive mathematical approach to modeling of enhancer functionality, it has the potential to unravel complex molecular mechanisms underlying transcriptional regulation.

We propose to investigate how dietary restrictions (DR) affect aging from a new angle by using social insects as models. Aging is a hallmark of most bilateria and most animals balance their reproduction rate against lifespan. Intriguingly, this trade-off is inverted in reproductive individuals (queens) of social insects (termites, ants, bees). Whereas most studies on aging directly manipulate the lifespan, e.g. of mice or worms or other lab-bred animals, we here propose a radically new approach by employing easily accessible and natural extremely long-lived termite queens as models. Their metabolism, response to DR and fertility will be gauged against genomically identical bu infertile and short lived workers, as well as shorter lived and less fecund queen of a closely related termite species. We will sample termite colonies directly from the field, keep them, expose them to DR and measure their fitness and fecundity. We will examine the role of DR during colony development by sampling transcriptomes, analyzing their epigenetic status, their metabolome and endocrine status and performing in-depth molecular analyses of key molecular components that are known to be implicated in regulating aging and fecundity. Using multiple OMICS methods, reverse genetics, hormonal and dietary administration we will be able to disentangle pathways involved in development of queens and measure the impact of energetic metabolic reprogramming on fitness and reproduction status. Expression patterns and spatio-temporal changes of genetic networks will be used to develop a simple state model. In this model, the metabolic status can be used to predict an individual's trajectory of aging and fecundity depending on its epigenetically imprinted background such as it's caste. Our project thus establishes a new model system for studying the relationship between DR, aging and fecundity, in which the latter two are decoupled and comparison of our model to other model organisms will help understand which dependencies and molecular components have universally conserved interaction partners or phenotypic effects. The project is possible only due to the four participants from three continents, with expertise in dietary research, energy metabolism, field research and social insect genomics.

Toxoplasma is a common parasite of birds, rodents, and humans during the asexual phase of its life cycle. But, having sex for Toxoplasma is limited to cat intestines. It enters cat when an infected rat or bird is eaten up. This creates an interesting problem because rats do not prefer to be eaten up. Toxoplasma blocks the fear of cats from infected rats. This is often taken to mean that infected rats will approach cat without fear and be eaten up at higher rates. We have no direct evidence for this indirect assumption. We will test this in the first part of this project. Kangaroo Island in Australia plans to fence off one of its parts and exterminate all cats from there. The island is doing so to protect wildlife and to reduce Toxoplasmosis in livestock. From our point of view, this is a golden opportunity to test if Toxoplasma increases predation of rats by cats. We can now compare the survival of infected rats and mice vis-à-vis uninfected rats and mice in the same landscape before and after cat removal. We expect to see more infected animals dying than uninfected when cats are around; and no difference when cats are removed.
While having sex is limited to cats, Toxoplasma can still sustain its asexual life cycle through carnivory or sexual reproduction. The sexual reproduction is exciting because such infections usually have less virulence. They do not kill a host very often as dead hosts do not reproduce to further transmit the parasite. Parasites moving across the food chain, from rats to cats for example, do not have such limitation and often increase virulence. But what happens when an infection is simultaneously transmitted by sexual and going-up-food chain mode? Both paths must trade-off with each other. When cats are plentiful it pays to increase predation (by reducing fear in infected rats) even if it means a bit less of sexual transmission and vice versa. This is a difficult assumption to test directly. The situation in Dudley Peninsula provides a perfect opportunity. We will collect Toxoplasma from bodies of infected rats and mice before and after cat extermination. We will bring these bugs in the lab and ask if cat removal means that the parasite now concentrates more on sexual transmission. That would mean a robust invasion of testes, more pheromones and more liking elicited from uninfected females. This is the second part of this project.

Organ development depends on the integration of local cell-to-cell interactions with long range signalling throughout the body. We will investigate mechanisms by which long-range signalling via the cerebrospinal fluid (CSF) regulate body axis formation and spine curvature. The CSF is produced by the choroid plexus in the brain ventricles and flows down the central canal in the spinal cord. The CSF instructs brain development by delivering age-dependent grow-promoting factors to target cells. CSF circulation also contributes to the curvature of body axis (embryo) and to the spine (juveniles). However, the mechanisms regulating the flow and content of CSF remain poorly understood. Progress has been impeded by a historical lack of tools and challenges inherent to studying fluids in small, developing organisms. The advent of innovative tools and approaches now provides an unprecedented opportunity to overcome previous limitations. The transparency of zebrafish and the accessibility to mouse CSF provide powerful models for testing our driving hypothesis that the physical and biochemical properties of CSF impact body axis formation and spine curvature. We propose an entirely new, interdisciplinary collaboration of three leaders in their field: (1) physicist Francois Gallaire (EPFL, Switzerland), expert in the theory & modelling of complex fluid dynamics; (2) developmental neurobiologist Maria Lehtinen (Boston Children’s, HMS, USA), expert in analysis of the choroid plexus-CSF system; (3) biophysicist Claire Wyart (ICM, France), expert in imaging and sensory physiology in the spinal cord. Our multi-tiered approach will unravel the principal parameters driving CSF composition and flow. We will map CSF flow in the developing fourth ventricle and central canal (Aim 1), elucidate mechanisms regulating protein secretion into the CSF (Aim 2), and investigate mechanisms of active transport of instructive signals along the anteroposterior axis controlling organogenesis (Aim 3). The proposed studies will transform this historically understudied area of neuroscience into a robust field spanning CSF-based signalling in brain and spine. As studies of paracrine signalling and fluid dynamics lag far behind cell-intrinsic studies of signalling, our techniques and concepts should provide a roadmap for future studies of fluid niches throughout the vertebrate body.

Biofilms are surface-adhered communities of bacterial cells embedded in a polymeric matrix, and they constitute a major form of bacterial life. Biofilms originate in one of two ways: by clonal proliferation from a single founder cell, or by aggregation of many migrating cells. What are the respective benefits and challenges of these two modes of biofilm growth? To answer this question, we need to understand the physical principles that underlie both modes of biofilm formation. Specifically, for clonal proliferation, it is unclear how cell-level processes, such as directional growth and division, cell adhesion, and heterogeneous matrix secretion, give rise to the evolving macroscopic organization and properties of the mature biofilm. For aggregation, the mechanics of how collective cell migration leads to a biofilm remains controversial. Here, under Prof. N. Wingreen’s supervision, I propose an interdisciplinary approach that will address biofilm formation in two such contrasting systems. Collaborating with Prof. B. Bassler, I will develop an active liquid crystal model to understand the growth and internal ordering of Vibrio cholerae biofilms. Working with Prof. J. Shaevitz, I will investigate whether the aggregation of Myxococcus xanthus constitutes a phase separation or a dewetting transition. Their research programs, respectively, provide single-cell resolution of developing biofilms and traction-force maps of collective cell migration. These data call for active matter models — first to elucidate general principles of self-organization that apply broadly across species, and second to understand the evolutionary selection of species-specific modes of biofilm growth.

The idea of combining man-made and natural systems has fascinated humans for centuries. However, despite the explosion of life science advances, the integration of electronic and biological systems remains woefully underdeveloped. To address this, we will apply principles of Synthetic Biology to develop biological cells controlled by electronic signals.

We propose to create synthetic cellular receptors selectively activated by electrochemically activated peptide. In order to endow the system with the ability to activate a selected ligand:receptor pair in the presence of other polypeptides, we propose to construct peptide-selective bio-electrodes. Electro activation of the peptides decreases their affinity for the electrode and leads to its relocation to the two component cellular receptor that utilizes intramolecular proteolysis to activate transcription of the reporter genes. Using the team’s expertise in protein, cellular and electronic engineering we will design an integrated system where the activation of the reporter expression can be both controlled and monitored using electrode array in a few or even in a single mammalian cell.

This proposed approach enables the construction of a potentially unlimited number of orthogonal electrode/ peptide/receptor systems that allow multichannel information transfer between computing devices and biological cells. While the first embodiment relies on the relatively slow gene expression readout, the same approach can be used to control practically any biochemical process in real time. Such synthetic signaling pathways capable of performing logical operations will exert complex and precise control on cellular biology, leading to a first generation of bioelectronic hybrids.

Organizing microtubules (MTs) into arrays is fundamental for many cellular functions and critically depends on MT nucleation by the gamma-Tubulin Ring Complex (gammaTuRC). MT Nucleation occurs from the centrosome as well as from dispersed non-centrosomal sites and is temporally regulated. Although the role of gammaTuRC in MT nucleation is widely accepted, the tools for measuring this activity are severely limited. Current methods rely on indirect readouts using MT plus end tracking proteins, based on the rationale that nucleation generates growing MT plus ends. However, polymerization of MT ends generated by severing enzymes or MT rescue can also result in growing plus ends and may be mis-interpreted as nucleation. To address this, I propose to develop a biosensor for the specific detection of gammaTuRC-dependent nucleation within the cells. I will explore several alternative approaches designed to detect interaction between gamma-tubulin molecules in the gammaTuRC and alpha-beta-tubulin oligomers, as it occurs during nucleation. I will use tagged gamma-tubulin in combination with chemical or protein-based probes to generate a fluorescence signal upon formation of the gammaTuRC/MT interface. The signal will be based on FRET, SPLIT-GFP fluorescence complementation, or aggregation-induced emission by a “turn on” chemical probe. I will then use the biosensor to characterize MT nucleation in cycling cells and in post-mitotic neurons differentiating in vitro. This work will provide unprecedented insights into gammaTuRC-dependent nucleation and its regulation in space and time, and will significantly advance our understanding of how cells assemble and remodel MT arrays.

The ability to associate sensory stimuli with appropriate emotional meaning while computing an optimal behavioral response is essential for survival. One key regulator of this emotional learning is the neurochemical dopamine (DA) acting in multiple brain areas such as the medial prefrontal cortex (mPFC) and basolateral amygdala (BLA). DA’s modulation of neural plasticity during emotional learning has traditionally been studied using electrophysiology in individual neurons, but how it occurs on the level of large neuronal networks (ensembles) remains unknown. To address this gap, I will investigate DA’s influence on dynamic ensemble activity within the mPFC and BLA during pavlovian fear conditioning, a classic model of emotional learning. Using in vivo calcium imaging and optogenetic control of DA release in mice, I will test DA’s role in establishing ensemble representations of conditioned stimuli. The long-term goals of this project are to (1) reveal key principles by which DA modulates neuronal ensemble coding and (2) lay the groundwork for future studies investigating how abnormal changes in ensemble coding relate to DA-sensitive anxiety disorders such as post-traumatic stress disorder.

The brain is a ceaseless worker, endlessly integrating the information it receives into a continuous stream of conscious thoughts. Interestingly, even falling asleep does not completely suppress conscious thoughts, as exemplified by dreams. This is all the more surprising since wakefulness and sleep differ so greatly in terms of neural activity and phenomenology. What is then the core neural substrate enabling the generation of conscious thoughts? I will focus here on spontaneous thought generation across wakefulness and sleep. Spontaneous thoughts will be here defined as conscious representations that are not triggered by a specific external event (e.g. an image, a word). Mind-wandering is a classical example of spontaneous thoughts during wakefulness, and dreams during sleep. My goal will be to identify the neural correlates of spontaneous thought generation, i.e. the minimal set of brain regions that are jointly sufficient to spontaneously produce conscious representations even in the absence of sensory stimulation. I will use both correlational and interventional approaches. By directly stimulating the human brain, I will seek to understand how brain activations constrain the content of thoughts. Comparing the neuronal correlates of spontaneous thoughts in wakefulness and sleep will be quite powerful because spontaneous thoughts arise without external stimuli, removing confounds due to sensory stimulations. Furthermore, the resulting neural correlates of spontaneous thoughts will, by construct, generalize across different conscious states, allowing us to isolate purer neural correlates of consciousness than previously proposed.

Over a 100 years ago, Élie Metchnikoff’s visionary work laid the foundations to our understanding regarding the importance of the gut microbiota for the physiology of the host. Recently, technological breakthroughs sparked a renaissance in the research of the human microbiome. While many observational studies identify clinically relevant correlations between the structure of the microbiome and states of disease, our true aim is to uncover the molecular mechanisms and functional roles of host-microbe interactions. Here I describe an innovative sequencing-based functional metagenomic approach, termed FunMet-Seq, which utilizes transcriptional profiling of single human cells towards the discovery and characterization of microbially produced effector molecules. To this end, a library of microbiome-derived DNA, extracted from stool samples, is heterologously expressed in E. coli. Next, culture broth from the library is sampled, sterilized, and used to treat genetically barcoded HEK293-TN cells. Finally, massively parallel transcriptional profiling of single cells enables to identify culture broth samples which contain an effector molecule, and to trace its origin to a specific clone in the array of metagenomic DNA. While the realization of the FunMet-Seq pipeline is an ambitious goal, it will catapult forward our ability to understand the molecular mechanisms underlying the he complex microbial ecology in the human body. Moreover, as the role of the microbiome in human physiology becomes evident, deciphering the interkingdom crosstalk within the human body is critical to reveal the etiology of microbiota-related diseases, and is expected to produce therapeutic applications.

Circadian rhythms are present in almost all light-sensitive organisms from cyanobacteria through plants, flies, and mammals. Human hormone levels and metabolism are tied to circadian rhythms and affect sleep and wakefulness timings. Disruption of the synchronous action of circadian clocks leads to several human diseases, mainly sleep and metabolic disorders such as non-24-hour sleep wake disorder and diabetes. Most known circadian clocks stem from a transcription-translation feedback loop. Protein and mRNA levels oscillate with a 24 hour period, matching the day-night cycle. An intriguing exception is circadian oscillations in cultured red blood cells (RBCs); since RBCs are anuclear, the circadian rhythm must be non-transcriptionally controlled. RBCs are a major blood component, so their circadian rhythm might affect some of the many metabolic signals carried by the blood stream. Furthermore, the RBC circadian rhythms in individuals with hematological disorders may be out of sync or even cause the disorders’ phenotypes. I aim to use methods that complement each other (e.g., proteomics, metabolomics, and interactomics) to investigate in depth the molecular mechanism of the RBC circadian rhythms, which will advance our understanding of the rhythm’s role in metabolism signaling and hematological disorders. This strategy is suitable for resolving this question since the circadian rhythm is non-transcriptionally controlled and not much is known about it. Revealing the mechanism of the RBC clock will be the first time a eukaryotic non-transcription-translation based circadian rhythm is characterized.

How does an animal perceive the world and execute the appropriate behavior? This task requires sensory organs such as the eye to detect features of the environment, then for the brain to integrate this information and produce a pattern of motion directed at a goal. However there is currently little information about how animals encode features from their natural environment. Indeed, most of our understanding about visual neural processing in the fish brain comes from the use of artificial stimuli that lack the complexity found in the natural visual environment. Fish are ideal for investigating visual processing because their diverse behaviors and compact brains are amenable to modern neuroscience technologies. How does the brain process information? Most current understanding of fish vision relies on the common lab model zebrafish (Danio rerio), but other species have likely evolved distinct solutions for visual processing based on environmental and behavioral differences. To bridge these knowledge gaps, we propose to thoroughly characterize the natural visual environments of two species, zebrafish and Astatotilapia burtoni (an African cichlid), by collecting a large dataset of natural underwater videos in India (zebrafish) and Lake Tanganyika, Zambia (cichlid). We will then use these videos in laboratory experiments with a custom-built immersive viewing arena to examine which visual features from the natural environment elicit behavioral responses in the two fish species. To examine brain responses to natural stimuli, we will transfer existing technology for recording brain activity in larval zebrafish to cichlids. We hypothesize that there will be species-specific brain function adaptations underlying several behavioral differences between these species in response to visual motion. We will then expand from studying basic visual features to analyzing the appearance of other fish during social behavior. What drives fish to school or fight? How does the surrounding environment influence how fish detect the presence of conspecifics and other fish? Our integrative and comparative approach will provide a novel perspective on how vertebrate visual brain areas evolved to encode and adapt to the natural world.

Adolescence is a unique transitional period marked by deficits in impulse control and behavioral inhibition, often leading to high-risk behaviors. It has long been believed that adolescent impulsivity is a product of a delay in the maturation of the brain circuit supporting decision-making, but due to technical limitations, this link remains elusive. While research has correlated adolescent changes in isolated brain regions with changes in decision-making, no study to date has probed how these brain regions act as functional circuits to modulate adolescence impulsivity. Here we will take advantage of recent technological advances to (1) investigate the pattern of brain circuit activity during live decision-making in adolescent and adult animals, and (2) determine the microcircuit dynamics underlying this behavior to identify the circuit basis of adolescent impulsivity. This will pave the way for future experiments in which we will manipulate circuit function in vivo to modulate adolescent impulsivity in behaving animals. Approaching behavior from the standpoint brain circuit maturation will consolidate a major conceptual and technical shift in systems neuroscience. This shift will shed light on the biological mechanisms underlying behavioral changes during early life and adolescence, but will also fuel a novel approach to the study of the etiology of mental and neurodevelopmental disorders with early onset, thereby informing novel, more effective circuit-specific therapeutic strategies.

Plant development and growth are linked to cellular shape changes, which are controlled by genetic programs but also by perception of environmental signals, including mechanical cues. While both genetic regulation and mechanical control of morphogenesis were studied independently, there is a need to explore how cellular shape-associated strain and stress can mechanically regulate gene expression during differentiation. In animals, mechanical stimuli are known effectors of differentiation. They involve propagation of mechanical forces through the cytoskeleton to the nucleus, leading to chromatin remodeling and modification of gene expression. Thus, nuclear envelope proteins that control nuclear shape and transmit forces to chromatin play a key role in rapid triggering of gene expression. In plants, less is known about mechanotransduction from cell surface to the nucleus.
Using a systems biology approach and an interdisciplinary network, we propose to investigate how mechanical cues affecting cellular shaping are sensed at the nuclear envelope to drive chromatin remodeling in Arabidopsis. We will sudy a unique cellular model, the single root hair in an epidermal tissue context, with well-defined morphogenetic programs linked to cytoskeleton and nuclear dynamics. We will analyze root hair formation and growth in WT and mutants affected in either root hair development or nuclear shape. Combining in vivo live imaging and micro-mechanical measurements (rheometry), we will evaluate mechanical properties of cells and nuclei during root hair development and their dependence on cytoskeleton and nuclear dynamics in relation to gene expression. We will also determine how these mechanical, structural and biological properties are modified when a controlled mechanical stress is applied to the root hair cell during development. Our data will highlight proteins involved in mechanosensing, and we will evaluate their interaction with the nuclear envelope network. Live imaging and rheometry data will be correlated to finite element modeling to estimate strain and stress in the system for predicting chromatin remodeling following cellular and nuclear shape changes.
Altogether, this will highlight the molecular networks involved in mechanosensing at the nucleo-cytoplamic interface and reveal how gene expression is robustly regulated during cellular morphogenesis in higher plants.

Melanin is a unique molecule synthesized by all domains of life (bacteria, archaea, and eukarya), but it is most commonly known as a UV screening pigment in humans and other animals. However, surprisingly little is known about the secondary or tertiary roles of the molecule. The recent discovery that melanin-containing fungi located at the ruptured Chernobyl reactor display enhanced growth in the presence of gamma radiation, which is usually thought to be harmful, has hinted at the diverse functions of the molecule. A defining feature of melanin is the chemical and structural variability found across and within microbial species. This feature along with the ability to broadly absorb over the UV and visible spectrum has led to the hypothesis that microbial cells could have evolved to capture and utilize this energy. Low resource environments such as those found in deserts are the most probable habitats in which these adaptations could exist. The main objective of this proposal is to assemble a diverse team of scientists to apply new single cell and single molecule technology to studying divergent biology in one of the most extreme environments on the planet, the Atacama Desert.
Here we test the hypothesis that melanin is a key player in producing biochemical energy in microorganisms that are exposed to high amounts of radiation in the UV range. As part of the project, our international team, consisting of engineers, biochemists and microbiologists, will carry out a comprehensive analysis of the microbes living in the Atacama Desert, one of earth’s most UV-irradiated regions. We will identify alternate functions of melanin using state-of-art technologies to 1) sort out and identify melanin producers; 2) sequence their DNA for melanin pathway analysis; 3) characterize how melanin is being synthesized; and 4) link melanin to the photochemical conversion of UV light. Since microbial communities and melanin represent mixed populations, technology is needed which can probe individual cells and molecules. Only once this is accomplished, can sub-populations within each group be thoroughly examined. The impact of understanding melanin not only extends the fundamental knowledge of a commonplace pigment, but also can also prove useful for searching for life on other UV-irradiated environments such those on other planets.

The cell cortex is a dynamic network of actin filaments (F-actin), myosin motors, and accessory proteins coupled to the cell membrane, which mediates both the maintenance and changes of animal cell shape. Cell shape is determined by a balance between resistance to mechanical perturbation and the mechanical stresses produced internally by the actomyosin cytoskeleton. The precise architecture of the F-actin network and the dynamics of F-actin assembly and disassembly determine the accumulation of myosin-generated stresses, resulting in diverse behaviors from orchestrating local pulsatile contractions to inducing cell-scale polarised flows. For instance, in xenopus oocytes and embryonic cells of echinoderms and frogs, positioning of the actomyosin cytokinetic ring is preceded by travelling waves of F-actin assembly and disassembly1. Similarly, local pulsatile actomyosin contractions lead to persistent constriction within the drosophila mesoderm2, or global polarization and flow of the cortex of the developing C. elegans embryo3. Thus, contractility often follows the maintenance of a dynamic but steady cytoskeleton. However, how the spatial and temporal balance of actomyosin contractility, F-actin architecture and biochemical signaling determine homeostasis or polarized flows in the cytoskeleton remains an outstanding question in molecular cell biology.
Here we propose a unique integration of ex vivo, in vitro and in silico studies to uncover the molecular design principles for cell polarization. Ex vivo, we will modulate protein-protein interactions and use optogenetic control of Rho and light inactivation control of myosin to determine the extent to which cortical stability entrains force generation to impact polarization in neutrophils. Guided by such experiments, we will spatiotemporally modulate protein-protein interactions within an in vitro model cytoskeleton to alter F-actin architecture, adhesion, and myosin activity. In silico, we will systematically vary these parameters to generate testable predictions for cortical stability versus polarization. We envisage that our integrated approach will decipher the general principles underlying the assembly, stability and dynamics of cytoskeletal structures and the regulation of eukaryotic cell shapes.

During infection with bacteria or viruses, our immune system becomes activated to fight these foreign invaders and thereby prevent us from getting ill. Dendritic cells are an important immune cell type that control this process. When they sense the presence of infection, they trigger an immune response against any simultaneously captured bacterial or viral molecules by displaying the molecules on their surface for immune cells to recognise. However, they can also capture normal “self” molecules from our organs during infection, and it is unclear how they focus the immune response upon the foreign invaders without triggering an inappropriate response against “self” molecules that could inflict damage upon our organs. As we currently have only a limited ability to detect and visualise self and foreign molecules on the dendritic cell surface during an infection, our understanding of this process is incomplete.
We propose a bio-engineering approach to address this problem. Nature has already designed a system for biological molecule detection on the dendritic cell surface: immune cells called T cells are exquisitely sensitive at recognising specific surface molecules on dendritic cells. We will leverage the key features of this interaction to develop novel staining materials that imitate the natural way that T cells recognize molecules on dendritic cells. Using an engineering technology based on DNA origami to control the size, shape, and function of small particles, a “synthetic T cell” staining reagent will be assembled on a DNA scaffold. By adding fluorescent signals and experimenting with a variety of engineering blueprints, we can increase the sensitivity and signal intensity of our particles. Combining these synthetic T-cells with state-of-the-art microscopy techniques that can image individual molecules, we will precisely pin-point the exact location of self and foreign molecules on the DC surface during infection to determine if their organisation helps to either inhibit or promote an immune response. Overall, this project will thus employ expertise from a range of different disciplines to dissect how the immune system discriminates between “self” and “foreign” molecules.