KinBioFRET proposal is based on an original combination of multidisciplinary partners: technological and methodological development team (Marc Tramier) directly concerned by the development of FRET biosensor methodologies and three biological partners (Jean-Pierre Tassan, Claude Prigent and Roland Le Borgne) directly interested into dynamics of kinase activities in different fields of biology. We propose to develop new FRET biosensors to measure the activity of two essential mitotic kinases, MELK and Aurora A, in the frame of spatio-temporal regulation of these kinases during cell division in living genetically tractable model organisms. First, to be able to increase sensitivity, speed of time-lapse acquisition and then biological relevance of these bioprobes, KinBioFRET will develop new technological and methodological approaches by using fastFLIM for quantitative heteroFRET measurement and by introducing homoFRET bioprobes by fluorescence anisotropy measurement. Second, one of the methodological objectives is to be able to follow in the same time two (or more) FRET bioprobes using the homoFRET approach. This will be achieved by measuring MELK and Vinculin tension sensors using multicolor homoFRET biosensors simultaneously in Xenopus embryos. Third, we will engineer new FRET biosensors to study spatio-temporal regulation of the mitotic kinase Aurora A in Drosophila. The choice of Aurora A is intimately linked to the biological questions addressed by four independent groups of biologists at IGDR. In addition to the FRET change from C- and N-ter fluorescent protein tags within Aurora A, we propose to develop an Aurora A FRET biosensor constituted of a phospho-binding domain recognizing an Aurora A substrate peptide sequence and sandwiched between two fluorescent proteins. This latter FRET biosensor will contain various target sequences for specific subcellular localizations such as centromers, centrosomes, and plasma membrane. Finally, the methodological approaches developed here will be directly used to investigate two biological questions: (i) the comparison of blastula and gastrula dividing cells to simultaneously follow spatio-temporal dynamics of MELK kinase activities and mechanical tension in Xenopus embryos, and (ii) the spatio-temporal regulation of Aurora A activity following symmetric and asymmetric cellular division in the context of Drosophila live pupae. We believe that this proposal is novel and ambitious for several reasons: multidisciplinary approaches, development of quantitative fluorescence microscopy methods dedicated to FRET bioprobes, development of new Aurora A kinase FRET biosensors, development of homoFRET biosensors by anisotropy measurements, development of simultaneous spatio-temporal dynamics of FRET biosensors, novelty of expected biological results in the field of kinase regulation during cell division in developing living organism.

Histone post-translational modifications (acetylation, methylation, phosphorylation, etc.) play an important role in all chromatin-based processes. Specific combinations of histone modifications can specify various functions downstream. This chromatin “language” is known as the "histone code". Studies on the structure and function of this code have been focused on its role in regulating gene expression. However, several experimental data (including ours) show that post-translational modifications of histones play an important role in major centromeric functions such as sister chromatid cohesion or chromosome segregation. Our project aims to decypher the centromere histone code and identify the functions associated with post-translational centromeric histone modifications. Two axes, divided into three tasks are envisaged: 1 / Identification of histone modifications at the centromere (task 1). Centromeric chromatin is characterized by the presence of a centromere specific variant of histone H3, CENP-A. Previous studies have shown that centromeres are made up of blocks of nucleosomes containing CENP-A alternating with blocks of nucleosomes containing histone H3. We intend to take advantage of the physical link between the CENP-A and H3 nucleosomes to purify centromeric chromatin. We will conduct Tandem Affinity Purification (TAP) experiments using a cell line expressing a “TAP-tagged” version of CENP-A. Chromatin digestion will be controlled in order to copurify adjacent centromeric H3 nucleomes. Individual histones will then be purified by FPLC and their post-translational modifications profile will be established using FT-MS (Fourier-Transform Mass Spectrometry), a recent performant mass spectrometry technique. The respective profiles in interphase and mitotic cells will be compared. Using this approach, we will to map centromeric post-translational histone modifications and ultimately produce tools to study their functions. 2 / Function of previously described centromeric histone modifications (tasks 2 and 3). We have obtained preliminary results that suggest a role of phosphorylation of CENP-A serine 7 (CENP-SA7) in sister chromatid cohesion. CENP-A is phosphorylated by Aurora B but our preliminary results suggest multiple roles for Aurora B in sister chromatid cohesion. We will construct a “Shokat” version of Aurora B in order to precisely assess the functions of Aurora B in sister chromatid cohesion. We have developed an antibody against CENP-A acetylated on lysine 9 (CENP-AK9). We found that CENP-AK9 is actively deacetylated but we do not know the function of this acetylation / deacetylation balance. Our first experiences show that some lysine 9 substitutions of CENP-A are lethal and we want to construct cell lines expressing mutated forms of CENP-A under the control of an inducible promoter in order to understand the role of lysine 9 in centromere functions. Finally, our previous study suggested a role for centromeric histone H3 lysine 4 (H3K4) dimethylation in sister chromatid cohesion. We have identified the methyl-transferase responsible for this modification at the centromere and we want to assess its role centromere functions. We will also conduct "peptide pull down” experiments with a H3K4 dimethyl peptide and purify the retained complexes by FPLC. Retained complexes will then be analyzed by mass spectrometry to identify their components and their roles in centromere functions will be investigated.

It is now generally admitted that effects of climate change are enhanced in polar areas. Because of the ice-dependent character of Arctic marine ecosystems, climate-induced changes in sea-ice cover are expected to lead to shifts in primary production (decrease of ice algal production, increase in phytoplankton and microphytobenthos production) and changes in sea water chemistry (lower salinity and pH, higher temperature). Those changes will have repercussions on the entire ecosystem functioning and carbon cycling, although it is yet unclear how benthic organisms will respond to those changes in food sources and environmental conditions. Traditionally, the study of the Arctic Ocean has mainly been carried out by countries surrounding the Arctic Ocean, i.e. Canada, USA, Norway, Denmark and Russia. However, since climate change is felt first and foremost in the Arctic, growing concerns for Arctic ecosystems and native populations living there have brought other countries to join research efforts. In particular France, which carries important polar activities in Antarctica, recently started to extend its polar marine ecosystem research efforts to the Arctic, with new projects and with the creation of an International Research Unit of the CNRS in Québec. Although recent Arctic ecosystem studies have focused on describing the present state of either the "pelagic" or "benthic" compartment, the link between those two compartments, the "pelagic-benthic" coupling has often been underestimated. Moreover very few studies have included experimental approach in order to predict future scenarios, while this knowledge is crucial if we are to understand possible future changes and create models. The overarching goal of this study is to investigate how climate-induced changes in biological (food sources) and environmental conditions will impact the Arctic benthos. This project will combine existing data, new field data, and a new experimental approach which will test various scenarios of food (i.e. high food quality, low food quality) and environmental parameters (pH, salinity, temperature) therefore improving understanding of present state Arctic coastal ecosystem function, and prediction of possible feedback scenarios of the ecosystem to changes in a less ice-rich Arctic due to climate warming. The work will be separated in 4 tasks: - Description in great details of the seasonal variability in pelagic-benthic coupling, combining both pelagic and benthic perspectives - Study experimentally the impact of changes in food quality for the benthos - Study experimentally the impact of changes in temperature, pH and salinity, on key bivalves species - Development and calibration of models of carbon and energy fluxes in the ecosystem and in the key bivalves species This project will take place at the French/German research station in Ny-Alesund, Svalbard, which will provide adequate set up and background data insuring its success. In addition to its scientific goals, this project aims to create international sustainable collaborations between France, Norway, Germany and Canada so experts can bring and share their expertise for a better understanding of the whole Arctic ecosystem functioning. In particular, this project will be held in tight collaboration with the French project Apolobis, aiming to establish an Arctic observatory based on bivalves in Ny-Alesund, and the Norwegian project Icicles, aiming to understand effect of climate change on key zooplanktonic organisms.

Asymmetric cell division is a conserved mechanism by which cell fate diversity is generated during Metazoan development. How one cell can generate two daughter cells with different identities and how defects in this asymmetry can contribute to pathologies are the fundamental questions we are addressing in Drosophila. We are investigating this process in the context of asymmetric cell division of neural precursor cells called Sensory Organ Precursor (SOP). These latter undergo four rounds of asymmetric division, in which mother cells generate distinct daughters via the unequal segregation of the cell-fate determinants Numb and Neuralized (Neur) at mitosis. At each division binary cell fate decision are regulated by Delta-Notch dependent cell-cell signalling. Notch receptor is activated by the ligand Delta present at the surface of adjacent signal-sending cell (trans-activation). Numb is an endocytic protein that can bind to Notch and Sanpodo (Spdo), a four pass transmembrane protein required for Notch activation in SOP lineage, thereby preventing Notch activation in the cells inheriting Numb. Neur acts in SOPs and pIIb cells, the signal-sending cells, to regulate the endocytosis and signalling activity of Delta, thereby promoting Notch activation. Despite intensive studies, the mechanism by which Neur regulates Delta activity is not known. The nature of the ligand activation and the subcellular localisation where recycled Delta could interact with Notch to produce signalling remained unknown. Similarly, endocytosis of Notch is proposed to play both positive and negative roles on the signaling. Our research aims to understand how intracellular trafficking regulates the spatio-temporal regulation of Notch-dependent fate decision. We have performed a genetic screen that led to the identification of novel potential regulators of Notch signaling. Among them, we identified the clathrin adaptor AP-1 complex. While mammalian AP-1 is involved in lysosome-related organelle biogenesis and in polarized sorting of membrane proteins to the basolateral plasma membrane, the function of Drosophila AP-1 was largely unknown. We reported that AP-1 localized at the trans-Golgi Network and in recycling endosomes, acts as a negative regulator of Notch signalling. Inactivation of AP-1 causes ligand-dependent activation of Notch leading to a fate transformation within sensory organs. Loss of AP-1 causes apical accumulation of the Notch activator Sanpodo and stabilization of both Sanpodo and Notch at the interface between SOP daughter cells, where DE-Cadherin is localized. Our data point towards a specific DE-Cadherin-rich junctional contact containing Notch and Sanpodo that could serve as a launching platform from where ligands are trafficked for signalling. While our results have begun to shed light on the function of AP-1 in the control of Notch signaling, several important issues remained unsolved. We here propose to investigate the molecular mechanisms by which AP1 complex regulate Notch signaling. To this aim, our research proposal is aimed at: 1- identifying the site and mechanism of action of AP-1 at both light and electron microscopy level. We propose to develop and adapt correlative light and electron microscopy to clonal analyses in Drosophila 2- characterizing novel interactors of AP-1 identified by genetic (our screen) and biochemical (collaboration with Pr. B. Hoflack, Dresden) means 3- investigating the link between AP-1-dependent E-Cadherin junctional domain and Notch signaling (launching platform). To reach this goal, we propose to combine Drosophila genetics to cutting edged cell biology approaches to investigate in molecular terms the respective role of regulators identified in the screens as well as the role of DE-Cadherin junctional domain in the regulation of Notch-dependent binary fate decisions following asymmetric cell division.