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.

This work leverages cutting-edge genomic and shape analysis methods to study the evolution of mammalian cooperative societies. Current approaches to studying vertebrate cooperative behavior work in the classical, but mechanism-free, frameworks of behavioral ecology and life history theory. Thus, while cooperation itself is of long-standing interest, we know little about how animals that occupy distinct roles in cooperative societies differ at the molecular level.
Here, we propose to integrate new molecular and computational approaches with a 24-year field study of the most cooperative nonhuman mammal yet described, the meerkat. Like cooperative insects, meerkat societies are characterized by a division of social roles: adults are dominant breeders or morphologically and physiologically distinct helpers, who feed and guard the breeders’ young. However, all helpers retain the capacity to transition to breeder throughout life. Meerkats thus present an exceptional opportunity to study alternative phenotypes in cooperative societies. We will first investigate how helpers and breeders are differentiated at the gene regulatory level, including whether steroid hormone signaling generates these differences. Second, we will test social role-driven differences in growth and immune defense. We will track growth by developing computational geometry-based approaches to perform 3D skeletal reconstructions from X-ray data and immune defense using experimental pathogen challenges. Finally, we will test the hypothesis that the competing demands of growth, reproduction, and immune defense create “competition” at the transcriptional level, which is resolved differently by helpers and breeders. Our experiments will not only quantify phenotypic transition-associated trade-offs, but also identify the genes and pathways that mediate them.
The methods required for these analyses either do not exist or will need to be generalized to field studies for the first time. Thus, this study requires collaboration across behavioral ecology, genomics, immunology, and computational image analysis. Together, it will contribute a powerful model for applying modern tools to long-standing puzzles in evolution and behavior. It will also yield new insight into the molecular changes involved in the evolution of cooperative societies—a subject of fascination and controversy for almost two centuries.

Pre-implantation development in mammals involves complex processes at the level of genomic organization, DNA methylation and transcriptional activity, leading to embryonic genome activation. How different epigenetic markers interact in single mouse blastomeres at early stages of development to influence the transcription of genes, and to what extent these processes vary from cell to cell and between embryos, is not fully understood. Although, in recent years a number of methods that enable single-cell omics measurement have been described, their integration to measure more than one entity in a single cell has remained challenging. Here, I propose a novel strategy for the integrated measurement of different epigenetic markers in combination with the measurement of nascent transcripts in single cells. The strategy relies on the use of a combination of specific restriction enzymes, for the measurement of 5-methylcytosine (MspJI), 5-hydroxymethylcytosine (AbaSI) and accessible chromatin (MseI and/or NlaIII), as well as 2’-fluoro GTP analogues in combination with specific RNases for the measurement of nascent transcripts. The application of these technologies to pre-implantation mouse embryos will shed light into the relationships of different epigenetic markers and their influence on transcription at the single cell level, and how these processes result in cell differentiation during early development. I further propose to use new deep-learning libraries, such as TensorFlow or Caffe, to quantitatively model and gain understanding of these dynamic processes in single blastomere cells.

The poor regenerative capacity of central nervous system (CNS) neurons leads to devastating and irreversible outcomes after injury or disease. The CNS is an immune privileged site, which maintains specialized crosstalk with the immune system. While the immune response to acute injury in the CNS is very similar to the wound healing process in other parts of the body, this response is oftentimes insufficient to achieve significant repair and restoration of neuronal function. One of the key challenges in analyzing this response is the diversity of the types of cells that participate in the process, which change with time and some of which may be unknown. Cutting-edge advances in massively parallel single-cell genomics, many pioneered by the Regev lab, will now enable me to analyze the entire injured tissue, at high, single-cell resolution, that takes on its complexity, heterogeneity, and temporal variation. Using this approach and comparing low- and high-regeneration states could help identify junctions where the immune response is insufficient to support regeneration, new cell types involved in the process, and targets for therapeutic intervention. Emerging cellular and molecular candidates will be perturbed experimentally in vitro and in vivo, using CRISPR/Cas9 technology and cell augmentation and depletion experiments, to validate their significance for neuronal regeneration. Human samples will be incorporated to establish validity in the clinical setting. The findings could be relevant for different types of CNS trauma, inflicted though mechanical or biochemical injury, autoimmune, neurodegenerative or vascular disease, at the brain, eye and spinal cord.

For individual genes, the number of transcripts per cell typically increases with cell size, yet how this is achieved is unknown. Recent image-based analysis suggests that both cell volume and surface area contribute to this regulation, with distinct classes of genes showing tighter correlations with either one of these two features. Here, I aim to go beyond these correlations – dissecting the mechanism by which cell geometry shapes the transcriptome. First, using quantitative RNA-sequencing and RNA metabolic labelling in cells with a range of defined shapes and sizes, I will define genome-wide classes of transcripts that scale with cell volume and surface area, and analyze the extent to which regulation of transcription and RNA stability contribute to this scaling. High-resolution mapping of changes in chromatin accessibility and modification state in these constrained cells will also be used to reveal the basis of transcriptional control and to suggest DNA-binding factors involved. Second, high-throughput image-based quantification of specific transcripts in single cells will be combined with genome-wide genetic screening. For each single-gene knockdown, this will generate a multivariate dataset containing the absolute abundance of a panel of selected transcripts in addition to hundreds of single-cell and population-context features. I will specifically identify and pursue genetic perturbations that disrupt the scaling of transcript abundance with either cell volume or area. Following these unbiased approaches with hypothesis-driven experiments and mathematical modeling will provide insight into how single cells precisely adapt genome expression to cell size and shape.

Human mobility is made possible by skeletal muscles, a voluntary tissue under the control of the central nervous system. Upon injury or insult, skeletal muscle regenerates to restore its form and function. Muscle regeneration requires an adult stem cell population termed satellite stem cells (SCs) that reside in a specialized ‘niche’ atop the sarcolemma of multinucleate myofibres and ensheathed by a proteinaceous matrix. Most of the time SCs are quiescent; a state characterized by inactivity. Injury induces SCs to activate and produce committed progenitors called myoblasts that fuse into multinucleate myofibres. In the present state of our knowledge, SC activation is an essential and intriguing process that is not well understood. Although SC activation is tightly controlled in the body, when SCs are removed from the body and studied in two-dimensional culture, activation is the default state. This observation points to the enticing possibility that physical or structural aspects of the three-dimensional tissue microenvironment might contribute to the spatial and temporal control of SC activation. In particular, we hypothesize that injury rapidly alters the SC niche mechanical stress profile and that this serves to engage intracellular mechanisms that tip the balance in favor of SC activation over quiescence by directly altering transcriptional activity in a highly localized manner. The Gilbert (Canada) and Betz (Germany) labs share a common interest in resolving the impact of mechanical stresses on cellular fate, and the Darzacq (USA) is driven by a curiosity as to how the nuclear environment imposes on the fundamental rules of gene regulation. By joining efforts in an international collaboration, we aim to (a) define the mechanical stresses (e.g. compressive, shear) exerted on SCs in resting and regenerating niches and (b) determine the molecular implications of niche mechanics as they relate to the SC transcriptome in the native niche, for the first time. We will integrate our three distinct experimental approaches – SC cell and molecular biology (Gilbert), quantitative biophysics (Betz), and high resolution single molecule imaging of transcription factor binding and nascent transcript activity (Darzacq), to conduct a collaborative study and achieve a holistic understanding of the earliest events that evoke SC activation in the native niche.

Sleep takes approximately one third of our lives yet its functions remain elusive. Extensive efforts have been made and hypotheses raised to link sleep with synaptic plasticity and memory consolidation. The sleep/wake pattern and sleep architecture evolve as development proceeds, and so does cortical connectivity, as featured by spinogenesis in early postnatal life and spine pruning during later adolescence. Several subcortical neuromodulators, including hypocretin, norepinephrine and dopamine, play critical roles in promoting arousal and thus control the sleep/wake cycle. We hypothesize that these arousal circuits help to shape the synaptic connections in cortex during development and the disruption of coherent sleep/wake cycle at certain developmental stages may cause defects in brain functions in adulthood. Mechanistically, synapses “tagged” by the arousal inputs during the previous wake phase may be selectively strengthened while other “untagged” synapses weakened in the following sleep. We will combine a series of state-of-art techniques including optogenetic and/or chemogenetic manipulations, viral tracing, two-photon live imaging as well as fiber photometry to interrogate these hypotheses, which will broaden and deepen our understanding towards sleep and brain plasticity in a developmental point of view and provide potential clinical insights on sleep and arousal disorders.

Living cells organize their interior in a non-random, polarized manner to carry out specialized biological functions. Cell polarity arises from a complex interplay between the plasma membrane, associated proteins and cytoplasmic molecules. In eukaryotic cells, symmetry is in most cases initially broken at the level of membrane-bound molecules such as phosphatidylinositol lipids and Rho-type GTPases. These conserved signaling hubs synergistically control cell morphogenesis and movement through the actin cytoskeleton. While many of the components of these intimately linked systems have been defined, very little is known about the biochemical mechanisms underlying cell polarity and shape changes. Instead of studying intracellular pattern formation and actin assembly in the complex cellular environment, I want to establish the minimal requirements for these processes by re-building them from purified components. To this aim, I will combine multiprotein reconstitution on artificial membranes with advanced fluorescence imaging techniques and employ synthetic biology methods. Specifically, I aim to understand (i) how membrane-associated signaling systems self-organize into macroscopic spatial patterns and (ii) how membrane polarity can be harnessed by the actin cytoskeleton to construct distinct structures at opposing ends of the cell. As a whole, this work has the potential to advance our understanding of the mechanistic foundations of cell polarity and morphogenesis at the systems biochemistry level.