A shared feature of many anxiety disorders is the overgeneralization of fear, the tendency to falsely link together events that have differing emotional significance. In the case of abnormal fear learning this can produce heightened levels of arousal and fear in situations that should be perceived as safe.
Previous studies indicate several distributed brain areas interacting with neuronal networks in the amygdala to modulate the fearful perception of diverse stimuli. It therefore remains largely unknown how changes in synaptic and cellular function (synaptic scale) can influence encoding of fearful stimuli in large cell networks (network scale) and how interaction between brain areas (global scale) finally leads to overgeneralized behavioral responses. Although a few studies have begun to examine neural substrates of fear generalization at the synaptic and network level, to date, no structured and integrated picture has been generated. This major gap exists because no study so far has been able to integrate experimental results across the different scales to generate a complete model of interactions that regulate fear generalization. As a consequence, new therapies and drugs to treat anxiety disorders emerge only slowly with no major discoveries in sight.
In this proposal, we aim to utilize a unique combination of expertise in neurophysiology, optogenetics and advanced brain imaging to study the mechanisms of fear generalization across spatial scales, ranging from synapses to individual neurons, to large-scale, distributed networks across the whole brain. For the first time we will investigate how local amygdalar networks regulate fear generalization within the context of their afferent signals emerging from global brain activity. To connect synaptic, network and global scales, we will develop new innovative technologies such as an fMRI compatible version of a miniaturized microscope for functional Ca2+ imaging to simultaneously monitor whole brain function and local network activity during fear generalization tests in mice. We will complement these studies with in vivo Ca2+ imaging, optogenetics, and ex vivo electrophysiology to identify the distributed inputs and local circuits in the amygdala that generate overgeneralized fear responses.
The goal of our interdisciplinary systems neuroscience/engineering approach is to provide a comprehensive understanding of the neural substrates that trigger overgeneralized fear, thereby generating a novel framework for how anxiety-related behaviors emerge across different brain scales.

In this project we will uncover the evolutionary dynamics in both humans and pathogens in response to epidemics. Recent technology advances enable detection of infectious agents in ancient DNA (aDNA) samples that tie to major historical epidemics. However, we know practically nothing about the dynamic process by which genetic adaptation occurs simultaneously in both the host and pathogen as a consequence of epidemic outbreaks.
We propose to integrate aDNA and sophisticated computational approaches to investigate the selective pressures imposed by the introduction of new pathogens during European colonization of the Americas. Our goals are to characterize the changes in both pathogen and human genetic diversity before and after European colonization to describe: 1) the genetic signatures that were putatively responsible for decimating the Native population, and 2) the selective and demographic processes that conferred adaptation to the colonization environment, especially with respect to pathogen exposure. To this end we will sequence the genomes of at least 30 individuals from before and immediately after colonization, and for the Colonial period we have access to archaeological remains of individuals who were likely victims of epidemics. We will also leverage the metagenomic data produced when sequencing aDNA and quantify the pathogen genetic diversity present in these samples before and after colonization. Lastly, we will use novel statistical methods to identify loci in post-colonization samples that depart from expected proportions of Native American, European or African ancestry to test whether admixture facilitated adaptation.
Our study will leverage temporal genomic data to address a long-standing question of how pathogens have influenced human evolution. As we lack studies quantifying jointly the changes in both pathogen and human diversity across time, this project offers a unique opportunity to directly assess, for the first time, how much evolutionary pressure is experienced within a human population when encountering new pathogens. Our design integrates novel paleogenomics approaches and cutting-edge methods development to leverage longitudinal sequence data of both ancient host and ancient pathogen sequence data to address coevolution with a temporal resolution that has not previously been reached.

Despite effort, it remains incredibly difficult to identify the cellular basis, and/or the causative genetic variants, underlying complex behavior. Understanding how behavior is encoded requires solving a dual problem involving both neurodevelopment and circuit function. Genes build nervous systems; nervous systems are activated to produce behavior. Streelman and Baier will collaborate to develop a unique model system to chart the complex path from genome to brain to behavior, in vertebrates from natural populations. In Lake Malawi, male cichlid fishes construct sand ‘bowers’ to attract females for mating. Bower building is an innate, repeatable natural behavior that we quantify in the lab. Males build two bower types: 1) pits, which are depressions in the sand, and 2) castles, which resemble miniature volcanoes. Species that build these two bower types can interbreed in the lab. Remarkably, first-generation hybrids of pit- and castle- species perform both behaviors in sequence, constructing first a pit and then a castle bower, indicating that a single brain containing two genomes can produce each behavior in succession. Moreover, brain gene expression in these hybrids is biased towards pit- alleles during pit digging, and castle- alleles during castle building. This phenomenon of allele-specific expression matched to behavior is compelling and offers the chance to identify the genome regulatory logic and neural circuitry underlying complex behavior. Streelman’s group will use single-cell RNA-sequencing to pinpoint specific cell populations that mediate context-dependent allele-specific expression in male bower builders. Baier’s team will use genome editing and optogenetic tools to manipulate the neurons that matter in the brains of behaving Malawi bower builders. Our collaborative work will thus identify the neurons responsible for biased allelic gene expression matched to behavior, and then manipulate those neurons to modify behavioral output. Achieving our goals will demonstrate how the genome is activated in particular cell types to produce context-dependent natural social behaviors.

Capturing the dynamic 3D atomic-scale structure of a macromolecular machine while it performs its biological function remains an outstanding goal of biology. Conventional structural tools (e.g. X-ray crystallography, NMR & cryoEM) only provide ‘snapshots’ of stable states along a reaction pathway. Reaction intermediates, and in particular short-lived intermediates, are hard to capture and characterize with such conventional techniques. Here we propose to combine (prior) information from multiple existing static structures of stable states with dynamic datasets of inter-atomic distances obtained by high-throughput non-equilibrium single-molecule FRET (smFRET) measurements in a microfluidic mixer using novel time-resolved multi-pixel single-photon avalanche diode detector. These measurements will be performed on libraries of molecular constructs, sampling multiple inter-atomic distances as function of reaction time. These measured distance distributions will then serve as multiple intra- and inter-domain distance constraints which, together with prior information (available structures), will enable the Rosetta software to achieve large-scale energy optimization-based refinement of time-resolved ‘snap shots’ of complex structures with improved accuracy. These time-resolved Rosetta structures together with intermediary molecular dynamics simulations will allow solving the 3D atomic-level structure of the macromolecule for each sampled reaction time point, eventually producing a 3D structural dynamic movie of the macromolecule in action. To demonstrate the utility of the proposed method, we will solve the dynamic structure of RNA polymerase during transcription initiation (promoter binding, bubble opening, abortive initiation, promoter clearance) and a pair of intrinsically disordered proteins (IDPs) involved in transcription regulation (ACTR and NCBD) that adopt a fully folded structure during a coupled folding and binding reaction. In addition to elucidating outstanding questions in transcription by combining detector developments, high-throughput and time-resolved out-of-equilibrium single-molecule FRET measurements with new experimentally-constrained molecular structure computational approaches, this multidisciplinary project will result in a new generic toolkit applicable to a large array of enzymes, proteins and molecular machines.

To understand how the brain represents the world is a central theme in neuroscience. Neural circuits encode information in terms of rate, timing and synchrony of action potentials arising from the activity of a complex spatial organization of excitatory/inhibitory neurons. The identity of the active neurons at any given time has a profound effect onto the final outcome and regulates fundamental human psychophysical behavior. Attempts to decode the complex behaviors in neurons has led to development of several neuromodulation and sensing tools, by which neural activity can be controlled by microelectrodes or through genetically altering specific neural circuits, ultimately to deliver electrical impulses. Unfortunately, these techniques introduce strong perturbations to the observed system, without reaching the desired spatio-temporal resolution. Experimental approaches that allow non-invasive activation of specific phenotypic groups of neurons could introduce systematic variation of the timing of signals revealing how neural ensembles encode information. Here we propose a novel (nano)tool to alter membrane potential in specific neuronal phenotypes in a spatiotemporally controlled manner.
The nFlare project will develop a novel research paradigm in neuroscience based on a new class of injectable nanodevices delivered to neurons and anchored to their membranes. These nanotools have the ability to depolarize cell membranes or to deliver active biomolecules. Electric or chemical stimulation of single cells will be achieved using deep-penetration near infrared excitation light, to reduce invasiveness.
nFlare core aims are: i) the development of nanodevice components able to generate photoelectrochemical current upon illumination with light; ii) state-of-the-art biochemical functionalization of the nanodevice to target specific neurons and to reduce inflammatory response; iii) applicability of such nanoscale devices as high spatio-temporal neuronal activity modulators in 2D and 3D in vitro neuronal networks followed by in vivo validation.
Selective modulation of neural circuits by artificial stimulation of neuronal membrane or controlled delivery of environmental factors could help answering fundamental neurobiological questions.

How cell differentiation and morphogenesis are coordinated during embryonic development is an open question in developmental biology. The accepted theory sees morphogenesis as a consequence of the cell differentiation events initiated by biochemical signals. However, recent evidence shows that embryonic cells can adjust their differentiation program according to the size and duration of their cell-cell contacts, suggesting that the interplay between biochemical signals and mechanical stimuli determines cell fate. Yet, how these stimuli are integrated in the gene regulatory networks of cell differentiation is largely unknown. Here I will pioneer an evolutionary approach to find conserved mechanisms by which cell-cell contacts regulate gene expression, by comparing two echinoderm species, sea urchin and sea star. In both species, mesendoderm differentiation depends on Wnt/ß-catenin signalling. However, they present different patterns of Wnt/ß-catenin activity and different cell-cell adhesion properties, with sea star cells being far less adhesive than sea urchin cells. Using a combination of live imaging, scRNAseq and theoretical modelling, I will determine how cell-cell contacts regulate mesendoderm cell differentiation in the two species and if variation in cell-cell contact formation underlies the divergence of tissue patterns between sea urchin and star embryos. This study will uncover basic principles of cell differentiation and tissue patterning, broadening our understanding of embryonic development and evolution.

Brain metastasis (BrM) represents the most common brain malignancy, predominantly arising from non-small cell lung cancer, breast cancer and melanoma. We hypothesize that the tumor microenvironment (TME) plays an essential role in the establishment and progression of BrM. The brain is constituted by a unique TME, which includes tissue-resident cell types such as astrocytes and microglia, and critically, the blood-brain barrier (BBB). Cancer cells are able to exploit the brain vasculature for their own benefit, by forming the blood-tumor barrier. This specialized vasculature is an essential TME compartment, as it may suppress anti-tumor responses by blocking immune cell infiltration, specifically of cytotoxic T cells. Indeed, a new perspective to target the tumor vasculature in combination with immunotherapy has recently emerged from studying other cancers; however, this concept has not been explored in BrM to date. Therefore, in the proposed project, I will investigate the biology of two major cellular components of the BBB: endothelial cells (ECs) and pericytes in BrM arising from distinct primary tumors. For this purpose, I will perform transcriptomic analysis of these cell types in patient BrM samples originating from lung, breast or melanoma primary tumors. I will combine this analysis with different experimental approaches available in the host lab, including immunocompetent mouse BrM models and in vitro and ex vivo cell cultures that have been optimized to model the brain TME. This integrated and complementary experimental strategy will facilitate the identification of innovative therapies for BrM to be used alone or in combination with immunotherapies.

Pain sensation, though unpleasant, is considered protective as it provokes aversive behavior from noxious stimuli. Studies show that sensory neurons, which generate the perception of pain and touch, interact with another important protection mechanism, the immune system. Ablation of sensory neurons can alter the skin-resident immune cell response. However, many aspects of this interaction remain uncharacterized.
Cells and antigens from barrier sites, like the skin, are drained and sampled by the lymph nodes (LN). Interestingly, LN are innervated by fibers expressing the sensory neuron derived-peptides Substance P and Calcitonin gene-related peptide. Some of these fibers are found in close proximity to leukocytes. However, their direct impact on immune cell activity is unknown.
This project aims to characterize the effects of sensory neurons in the skin and LN on immune cell behavior. To this aim, we propose to combine chemogenetics to locally activate sensory neurons, with a broad analysis of immune cell responses. Mice expressing Cre under the control of sensory neuronal markers, including the broadly expressed gene Advillin and genes selective for subsets of sensory neurons, will be injected intradermally or in the LN with a viral vector encoding the Cre-dependent expression of the hM3D(Gq) activating receptor. Once validated, this technique will be used to locally activate sensory neurons and characterize subsequent immunological changes using flow cytometry, histological and live imaging techniques. This project can provide new insights into how sensory innervations, which regulate behavior by creating the sensation of touch and pain, shape local immune responses.

A key brain region involved in accurate temporal refinement of motor and also cognitive behaviours is the cerebellum. Cerebellar granule cells (GCs) receive multisensory information from outside the cerebellum via mossy fibres (MF). The prevailing hypothesis is that, in order to learn temporal sequences, the cerebellum requires a rich diversity of GC activity patterns that as a population represent a distributed temporal representation of sensory inputs. But this has never been directly demonstrated. Unpublished network models from the hosting laboratory strongly suggest that their previous findings (input specific diversity of MF-GC synaptic dynamics) are sufficient to generate a diverse GC firing patterns that act as a temporal basis for cerebellar learning. We will use novel high speed two-photon in vivo imaging of GC firing patterns in behaving animals, and examine whether the diversity of GC firing patterns requires functional diversity of MF-GC synapses, and whether this mechanism is specific for different sensory modalities, using optogenetic activation of specific input types. Finally, we will optogenetically inhibit specific MF types to show their influence over specific timescales of eyeblink conditioning, then image the temporal representation in the GC layer before, during and after a temporal learning task. Expected results will included identification of the underlying cellular and circuit mechanism of temporal learning required for fine tuning motor and cognitive processes.

Pancreatic ductal adenocarcinoma (PDAC) remains an incurable disease with a high death rate. PDACs are exceptionally difficult to treat because of high genetic plasticity and encapsulation by cancer-associated fibroblasts (CAFs). Either are due to an altered epigenetic landscape where PDACs and CAFs show a switch towards global chromatin hypo-methylation, and concomitant hyper-methylation of tumor-suppressor genes. As CAFs do not show any mutagenesis, the molecular cause must be found in the tumor microenvironment.

The nutrient microenvironment represents a novel but important aspects of tumor biology. Nutrients are converted into the basic building blocks that fuel cellular proliferation, but also regulate epigenetic modifications. S-Adenosyl-Methionine (SAM) represents a nodal point in one carbon metabolism and is required for methylation of histones and DNA. We therefore propose that metabolism cannot sustain the high SAM requirement of both PDAC and CAF, thus shunting away carbons from epigenetic maintenance. This project therefore aims to map the link between nutrient availability, one carbon metabolism, availability of SAM, and the methylation profile of chromatin. Furthermore, we will establish a genetic sensor and CRISPR-based screening system to visualize the regulation of SAM compartmentalization, and attempt to normalize the epigenome of PDACs and CAFs through engineering of SAM metabolism (EpiSAMe). These studies will shed new insight into the crosstalk between metabolism and epigenetics in PDAC and provide novel molecular for the treatment of this devastating disease.

Genomic instability is a common feature of cancers and can have an important impact on cancer progression and response to treatment. However, the mechanisms underlying cancer genomic instability are not completely understood. While DNA repair defects are thought to be a major cause of this trait, other factors seem to be involved. In the last decade, metabolism has received a lot of attention within the cancer research community. Most of the work has focused on understanding the metabolic requirements of cancer cells for survival and proliferation. However, metabolism does not only provide building blocks for biosynthesis, but it also generates reactive by-products that can damage cellular components such as DNA. Thus, the metabolic alterations characteristic of cancer cells might not only support cancer development by supporting biosynthesis but also by promoting genomic instability.

The aim of this research proposal is to identify changes in the metabolism of endogenous genotoxins in cancer and to elucidate the role of these alterations, particularly in the context of genomic instability. In addition, the potential exploitation of this aspect of metabolism for cancer therapy will be explored. To achieve these goals a diverse range of techniques and study systems will be used, including transcriptomics, metabolomics, CRISPR-based genetic screens, cancer cell lines and mouse models.

In 2014, the Nobel Prize in Physiology was awarded for the discovery of place and grid cells that process spatial cues in the mammalian hippocampal formation;, the key structure for both navigation and episodic memory1. Place and grid cells, and additional cell types form the central building blocks in the current circuit models of navigation in mammals. However, a cohesive picture of how these circuits compute space and enable navigation has not been achieved. In fact, emerging evidence suggest that these navigation circuits are highly diverse in cellular phenotypes and functionality; they do not only map aspects of space but also elements like sound, time, and reward. How neural systems of spatial cognition have evolved outside of the mammalian clade is not clear, and comparative studies are critically needed to gain insights to basic functional constraints and structural requirements underlying these neural circuits.
The international research team of four PIs proposes a broad comparative systems neurobiological approach using teleost fish for integrative studies on higher navigation circuits. These fish are ideal models because they have conquered diverse spatial ecologies and show highly specialized sensory adaptions. Also, their brains exhibit an overall lower complexity to mammals, and are highly accessible to experimental manipulation. To establish systematic research on teleostean spatial cognition, the project combines neuroethological, electrophysiological, neuroimaging, and computational methodologies. Introducing a powerful electrophysiological recording technology in freely-moving fish and generating long-needed anatomical atlas resources, the project analyzes four teleost species with differing spatial ecologies. The team will uncover how different sensory modalities like vision, perception of depth, and active electrolocation are integrated during spatial navigation tasks, thereby investigating how top-down mechanisms modulate sensory integration of spatial learning.
Finally, the team will test specific hypotheses developed in small-scaled laboratory setups in an unconstrained natural environment. Here, the group will measure the activity of neurons in freely moving fish that explore a coral reef habitat. This will be the first ever attempt to analyze brain activity underlying navigation in the wild. Altogether, the project will provide new perspectives on the evolution, function, and mechanism of memory systems in animal navigation.

Prochlorococcus, the smallest photosynthetic organism, has diversified into many ‘ecotypes’ with finely tuned distinct ecological niches. The small and variable genomes of these cyanobacteria encode genes unique to particular habitats. A driving force for gene transfer and evolution in cyanobacteria is phage-mediated. While numerous reports describe lytic cyanophages that infect Prochlorococcus, surprisingly there is only indirect evidence that lysogenic phages – those that spend a portion of their life cycle incorporated into the host genome – exist. The central goals of this proposal are i) to analyze the occurrence of lysogens in wild populations of Prochlorococcus, and ii) to establish a robust protocol for gene editing allowing precise manipulation of the Prochlorococcus genome. The second goal will exploit, but is not dependent upon, advances from the first.
This will be the first study to assess lysogeny across natural Prochlorococcus populations and to develop phage-mediated genetic systems for engineering cyanobacteria. The development of efficient tools for genome editing would be extremely beneficial for understanding Prochlorococcus metabolism. It would allow functional studies for thousands of unannotated genes that influence the relative fitness of different ecotypes in different environments. A unique aspect of my proposal is the employment of a novel synthetic biology strategy to build engineered phages with broad host specificity, and to use them as vehicles for genome editing systems such as CRISPR/Cas9. This technique will be useful for several applications and will further establish Prochlorococcus as a model organism in environmental microbiology.

Phosphatidyl-inositol 3,4,5 triphosphoshate-dependent Rac exchanger (P-Rex) are a group of proteins involved in the activation of the small GTPase Rac, by acting as a guanidine exchange factor (GEF). Both Rac and P-Rex proteins are known to be overexpressed and altered in several cancers, consistent with their role in both cell mobility and their numerous interactions with both the mTOR axis and the PI3K/PTEN pathway. P-Rex1/2 specifically, are known to interact with mTOR and PTEN, among others, placing them at the nexus of regulatory pathways for cell metabolism, proliferation and mobility.
Despite their central role in cell signaling, very little is known about the interplay between such interactions. No structural information is available for the full-length P-Rex proteins and how different interaction partners affect its structure and function. I will therefore set out to better characterize the emerging properties of the multidomain P-Rex proteins by purifying both P-Rex1/2 and determining their structure using cryo-EM and orthogonal biophysical characterization methods. I then aim to define the interaction between P-Rex proteins and its regulatory partners, PTEN and the mTOR complexes 1 and 2. I will define their domains of interactions and determine the structure of the complexes, using a combination of cryo-EM and cross-linking and mass-spectrometry.
I expect to provide new insights into the functions of P-Rex proteins and their crosstalk with core cellular signaling pathways. Structures of full-lengths P-Rex alone or in complex with its interaction partners will serve as a platform for drug discovery, thereby providing new cancer therapeutic avenues.

The brain comprises billions of neural cells interwoven in a highly precise way and it is ultimately responsible for how we perceive, react to, and remember the world around us. We are fascinated by how the intricate structure of the brain is assembled step-by-step at an early stage of life (“embryonic development”), starting as a relatively shapeless mass within the nascent embryo and culminating in a highly complex organ in an adult. Specifically, we hypothesize that blood vessels that permeate the early brain have important roles in fostering brain assembly, and we further predict that they produce important cues that actively shape a specific early step of brain development. This is of great significance, because blood vessels were once thought only to passively supply oxygen and nutrients to various organs. We instead suggest an active role in which blood vessels function as a ‘signaling center’ to actively control the arrangement of neural cells during brain assembly.
We have assembled an intercontinental team to tackle this question via a three-part, integrated effort. First, we will create a map of where and when arteries and veins (two major types of blood vessels) first enter the nascent brain. Second, we will intermingle neural and blood vessel cells in a Petri dish and study their interactions. Third, we will block specific functions exerted by blood vessel cells and propose that this will have a commensurate impact on the spatial arrangement of nearby neural cells. These activities are innately highly collaborative and will involve interactions between the partner laboratories in each nation.
Taken together, we propose to explore how the brain is assembled during embryonic development and we will test whether blood vessels act not as passive support for, but rather as active executors of, this intricate process at early steps. If true, this has important ramifications. Not only will it shed light on how the early brain is assembled, but it adds to the emerging idea that neural cells and blood vessels share an intimate functional and spatial relationship, starting from the embryo and lasting into the adult — an idea pertinent to early brain assembly; our efforts to engineer this process in a Petri dish for regenerative medicine; and finally, the origin of certain neurological disorders that are caused in part by blood vessel dysfunction.

Sporadically, novel and potentially devastating pandemic influenza A viruses (IAVs) are generated through genome reassortment between human and animal co-infecting IAVs. Such pandemic viruses emerge as a consequence of the segmentation of IAVs genome into a bundle made of 8 distinct viral RNAs (vRNAs). However the molecular mechanisms of vRNA intracellular transport and assembly into vRNAs bundles, which are critical for reassortment, remain largely unknown. Our project aims to elucidate these fundamental aspects of IAV life cycle by developing innovative approaches.
We challenge the original model that newly synthesized vRNAs, in the form of viral ribonucleoproteins (vRNPs), are transported across the cytoplasm on Rab11-dependent recycling endosomes. Based on our recent work, we hypothesize that the concomitant transport and assembly of vRNPs is driven by their physical association with remodelled endoplasmic reticulum (ER) membranes and Rab11-dependent transport vesicles distinct from recycling endosomes.
We will set up a cellular system which resembles the natural respiratory tissue targeted by IAVs, while being amenable to simultaneous imaging of the endogenous Rab11 protein and tagged vRNAs. We will develop two cutting-edge and complementary imaging methods: dual-color single molecule fluorescence in situ hybridization (FISH) in live cells for the tracking of distinct vRNAs that diffuse concomitantly, and cryo-Focused Ion Beam combined with electron microscopy in situ hybridization (EMISH) to image individual vRNPs and their transport vesicles at molecular resolution. We will further assess the role of cellular ER-shaping proteins by performing CRISPR/Cas9-mediated knockdowns, and by monitoring changes in the viral-induced remodelling of ER and biogenesis of vRNP transport vesicles by live fluorescence imaging and cellular EM.
The proposed research will require the very close collaboration between three partners with distinct but complementary expertise. The approaches developed jointly are poised to revolutionize our understanding of IAV multi-RNA genome transport and bundling, and thus help in the broader goal of achieving better prevention and treatment of influenza disease. Additionally, the proposed technical developments in live cell FISH and cellular EM, will have impact on other fields of studies well beyond the scope of the proposed project.

Glycolysis is a fundamental energy metabolic pathway which consists of ten enzymatic steps. Unlike the mitochondrion, which is a membrane-bound organelle, glycolytic enzymes are soluble proteins in the cytosol. Based on biochemical evidence, glycolytic enzymes have long been hypothesized to form functional complexes to sustain the rates of glycolysis. This purported complex, called the glycolytic metabolon, was a subject of intense study and debate thirty years ago. Still today we do not yet understand how these purported complexes are organized in cells to sustain local energy metabolism, or their physiological importance. This gap in knowledge results from the fact that when glycolysis was being rigorously examined forty years ago, the techniques did not exist to conclusively answer these questions. The main challenge in addressing these questions lay then, as now, in the ability to both examine the localization of the glycolytic enzymes in living cells, while understanding the biophysical and biochemical mechanisms of their association, and its implications in cellular physiology. We have established a collaboration to address these fundamental questions by making use of our joint expertise in vitro reconstitution and C.elegans physiology, using the energy demands of the C.elegans synapse as a model system.

Plants live in an ever-changing environment which is not always compatible with their survival. A major life threatening condition is drought stress. While most plants can deploy an array of physiological countermeasures to endure remarkable levels of stress, there is a huge variability in the different strategies that plants choose to adopt to deal with drought stress. Many plants evade detrimental stress conditions by activating their reproductive development (flowering) earlier compared to non-stress conditions, a strategy known as drought escape (DE). Notably, even in the most water-rich environments plants face unpredictable dry periods, yet how this information affects the floral network at the molecular levels is unknown.
We will address this question with a blend of molecular and genetics-based approaches. We will leverage known mutants with altered DE to identify candidate mechanisms that are drought sensitive and can act as molecular switch to activate flowering. To comprehensively define DE mechanisms utilized under natural conditions we will assess the variability of the DE response in natural plant populations and in crops, which were selected by human intervention for the different field scenarios. Our targeted large-scale screens will allow us to identify naturally occurring variants in the DE process and decipher the molecular mechanism responsible for DE activation. This information will provide breakthroughs in our understanding of novel regulatory mechanisms that play a role in driving developmental adaptations across extremely variable environmental conditions, their natural genetic variation and the selective forces that maintain such variation in populations, an important aspect for predicting and dealing with the effects of climate change. Finally, because flowering time is a major component of yield potential in crops, the defined mechanisms will help us develop breeding strategies targeted for sub-optimal irrigation scenarios to produce crops with ameliorated performances under drought conditions.

The endocannabinoid system (ECS) is involved in a variety of brain functions, including reward and aversion, mainly through type-1 cannabinoid receptors (CB1). The Nucleus Accumbens (NAc) is a key brain region in the control of positive and negative reinforcement. ECS impairments in the NAc lead to negative emotional states. By responding to neurotransmitters with intracellular Ca2+ increases and releasing gliotransmitters that modulate synaptic transmission and animal behavior, astrocytes play active roles in neural information processing. While CB1-mediated astrocyte-neuron communication has been shown in several brain regions, it is unknown whether this communication exists in in the NAc. Mitochondrial CB1 receptors (mtCB1) directly regulate mitochondrial energetic activity, synaptic transmission and behavior, but the role of astroglial mtCB1 in these processes is unknown. Combining electrophysiology, subcellular-specific live Ca2+ imaging and behavior, this proposal aims at defining the contribution of astrocytic CB1 (plasmatic and/or mitochondrial) to synaptic plasticity in the NAc and their behavioral impact. The specific goals of this proposal are to define 1) whether astrocytes in the NAc express functional CB1; 2) how astrocytic CB1 regulate synaptic transmission and plasticity in the NAc; 3) how astrocytic CB1 impact reward and aversion and 4) how astrocytic mtCB1 influence astrocyte activity, synaptic plasticity in the NAc and behavior. The expected results will shed light onto the involvement of astrocytes in brain motivational systems through the ECS, and would reveal astrocytes as potential targets for treatment of motivational disorders.

Compartmentalization into functional units is the key principle of cellular life. In addition to conventional membrane-bound organelles, cells utilize membrane-less biomolecular condensates to locally concentrate proteins and nucleic acids. Prominent examples of such condensates are nucleoli, P granules or centrosomes, which play central roles in diverse cellular functions. Recent studies have demonstrated that membrane-less condensates assemble by liquid-liquid phase separation of proteins that are characterized by multi-valency, intrinsic disorder and low complexity sequences. The molecular mechanisms that control assembly and disassembly, shape and size of such membrane-less organelles remain key unanswered questions of modern cell biology. To address this fundamental gap in our knowledge, I propose an interdisciplinary research programme that combines powerful in vitro reconstitution with systematic screening technologies. Based on my discovery that the chromosome surface protein Ki-67 acts as a surface-active agent (surfactant) for one of the largest membrane-less assemblies, the mitotic chromosome, I will explore the radically new concept of surfactants for the global regulation of other membrane-less organelles. Starting from defining the molecular properties, mechanisms and regulatory basis for the action of Ki-67, I will systematically identify and characterize the surfactants of other membrane-less organelles in human cells. This ambitious research programme has the potential to provide novel paradigms of spatial and temporal control of membrane-less cellular assemblies and thereby substantially advance our understanding of cellular organization.