Overview: Understanding of the neural circuitry that underlies animal behavior is one of the greatest challenges facing biologists. Progress in this area requires both the assessment of quantifiable and reproducible behaviors, as well as detailed knowledge of neuronal connectivity and the dynamic properties of the neurons themselves. This later requirement - detailed knowledge of connectivity and neuron properties- is a daunting challenge even the simplest vertebrates, such as the zebrafish (~10,000,000 neurons). Our most complete and quantitative understanding of circuits driving behaviors come from the simplest nervous systems such as those of C. elegans and larval Drosophila. Here, an international collaborative team from three research institutions will focus their efforts on a uniquely positioned experimental animal for the quantitative investigation and modeling of circuits and behaviors - the tunicate Ciona intestinalis, one of the closest relatives of the vertebrates. The Ciona larval nervous system, is remarkable in two ways: it shows unmistakable anatomical, genetic, and functional homology to those of vertebrates, yet is very simple, with only 177 central nervous system neurons. As starting points for this proposal, the team has in hand a complete connectome (and a second on the way), a classification of the CNS neurons into 25 functional groups, quantitative assays for range of larval behaviors, and circuit models for those behaviors. In this project we will derive single-cell transcriptomic profiles for cells and cell-classes identified in the connectome, and integrate and experimentally validate this new knowledge in quantitative circuit models of Ciona behavior. Finally, we will create a web portal for Ciona larval connectomic data, something that is currently lacking and limiting the accessibility of this model system to the wider scientific community. Intellectual Merit: While the behavior of tunicate larvae has been studied for many decades, the recent publication of the Ciona larval connectome provides a unique opportunity for major advances. In terms of nervous system complexity, Ciona is most comparable to C. elegans, but fundamental differences in the underlying neurophysiology of these two very distantly related organisms highlights how investigation of both will be complementary. For example, Ciona neurons are known to have sodium-driven action potentials, unlike C. elegans, and Ciona has sensorimotor modalities not found in C. elegans, including photoreceptor-driven visuomotor responses, and a gravitaxis response driven by a well-described otolith organ. Broader Impacts:Impacts to Scientific Community: Despite the research opportunities made available by the Ciona connectome it remains relatively unexploited. We will increase the visibility and accessibility of the existing connectomics data, and new data collected as part of this project, by creating an internet-based resource within the highly successful ANISEED web portal. We expect this web portal to be of value beyond Ciona researchers to data miners, circuits modelers with diverse interests and researchers with particular interests in comparative neurobiology. Impacts to the society: The project will provide training and mentorship at the undergraduate, postdoctoral, and assistant professor levels. All three PIs are committed to increasing the participation of traditionally underrepresented groups in science careers. The inclusion of undergraduates in this project will help to ensure the continued pipeline of underrepresented students entering research careers. We will also undertake outreach efforts targeted to K-12 education. Finally, this project will further international scientific cooperation.

SCIENTIFIC CONTEXT. Unicellular organisms need to adapt to rapid and unanticipated changes in their environment. Spores ensure their survival by encapsulating the genome in a protective configuration while awaiting optimal growth conditions. Indeed, yeast spores enter into a quiescent state with minimal metabolic activity and are surrounded by a thick protective wall. Yet, protecting the integrity of the genome in these conditions involves not only a dramatic decrease in transcriptional activity, an extreme nuclear compaction, but also the capacity to completely revert these processes to allow germination. The mechanisms involved in the establishment of genomic quiescence in spores, the protection of their genome and its reactivation, remain unknown. OBJECTIVE. Several lines of evidence show that chromatin is highly compacted in spores. In addition, histone H4 is hyperacetylated and this modification is essential for spore viability. This observation seems counter-intuitive because H4 acetylation (H4ac) is usually associated with transcription activation and open chromatin. Therefore, the general objectives of this project are to understand (i) the mechanisms by which H4 is hyperacetylated in spores, (ii) how H4ac is compatible with quiescence in spores and their chromatin compaction, (iii) whether and how H4ac prepares genome reactivation observed during early germination. IMPACT. Through EpiSpores, we will improve our general knowledge on H4ac signalling pathways, chromatin organisation and transcription regulation. Furthermore, yeast spores provide an alternative model system to investigate the molecular mechanisms of quiescence entry, maintenance and exit in all eukaryotic cells. Finally, our previous work on yeast spores has been translated to the treatment of fungal infections, collectively responsible for 1.5 millions of deaths per year. Our future work exploring chromatin signalling pathways in Candida albicans and their functional role in the virulence of this pathogenic yeast will be based on the technological development of this proposal. CONSORTIUM. This project brings together young researchers, by academic standards, with collective and synergetic expertise in yeast biology, genetics, biochemistry, interactomics, high-throughput genetics, super-resolution microscopy and epigenomic approaches.

Endocytosis is the general mechanism by which cells internalize micronutrients and cell surface receptors. Importantly, viruses, toxins, and bacteria often hijack the endocytic machinery to access the host cell. Increasing evidence points out that unconventional endocytic mechanisms exist parallel to the clathrin pathway, which is considered the canonical pathway in eukaryotic cells. However, their exact molecular mechanisms and function are not well understood, especially in the pathological context. The objective of FLOTAKE is to decipher the molecular mechanisms that drive flotillin-mediated endocytosis in physiology and during viral uptake, from the single molecule to the whole-cell level. In FLOTAKE, we will apply state-of-the-art techniques, such as optogenetics combined with super-resolution and correlative light AFM microscopy to decipher flotillin-mediated endocytic vesicle formation in physiology and in the context of emerging virus infection, such as the chikungunya virus (CHIKV). The outcome of this project opens the door to the identification of new endocytic processes leading to CHKV entry and that could be targeted for future development of innovative selective inhibitors with applications as antiviral strategies.

Successful embryogenesis requires the differentiation of the correct cell types, in defined numbers and in appropriate positions. In most cases, decisions taken by individual cells are instructed by signals emitted by their neighbours. A surprisingly small set of signalling pathways is used for this purpose. The FGF/Ras/ERK pathway is one of these and mutations in some of its individual components cause a class of human developmental syndromes, the RASopathies. Our current knowledge of this pathway is, however, mostly static. We lack an integrated understanding of its spatio-temporal dynamics and we can imperfectly explain its highly non-linear (switch-like) response to a graded increase in input stimulus. This systems biology project combines advanced quantitative live imaging, pharmacological/optogenetics perturbations and computational modelling to address, in an original animal model organism, 3 major unanswered questions, each corresponding to a specific aim of the proposal: ? Aim 1: What is the spatio-temporal dynamic of intracellular signal transduction in response to FGF during embryogenesis? ? Aim 2: How is the switch-like response to graded extracellular signals controlled at the molecular level? ? Aim 3: Can the results be integrated into a predictive computational model of the pathway? Through this approach, in a simple model organism, we hope to gain an integrated molecular understanding of the spatio-temporal dynamics of this pathway and of its robustness to parameter variations.

Mitosis is a fundamental process required for the generation of multicellular organisms, for tissue renewal and homeostasis. During development, both the orientation of the division plane and the timing of mitotic entry, have fundamental influence on the positioning of daughter cells and their organization into tissues. However, the mechanisms that control the timing of mitotic entry remain poorly understood. Entry into mitosis is triggered by the activation of a mitotic kinase cascade and the simultaneous inactivation of counteracting phosphatases. Since the mitotic kinases themselves are activated by phosphorylation, a central question arises: how are mitotic kinases activated while phosphatases activity predominates? During development, how does the regulation and cross-talk between mitotic kinases and opposing phosphatases ensure timely mitotic entry? The objective of this proposal is to decipher how the parallel regulation of kinases and phosphatases control asynchronous mitotic entry during development.