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.

One of the hallmarks of eukaryotic cells is their internal membrane compartmentalization, fundamental to normal cell function. Many human diseases result from deregulation of pathways linking membrane-bound organelles. The best-studied mechanism of communication between organelles is vesicle-mediated trafficking. However, recently we have begun to appreciate the significance of information transfer between intracellular organelles at membrane contact sites (MCS). MCS are zones where heterologous membranes, usually the endoplasmic reticulum (ER) plus a partner organelle, come into close apposition. These sites of intimate contact are places where lipids and small molecules such as calcium are exchanged. However, MCS are very poorly understood because so few of their components have been identified so far. Moreover, our understanding of the function of lipid trafficking at MCS, and how it is integrated into other cellular communication systems, is in its infancy. Contacts between the ER and PM are particularly intriguing, as they affect PM signaling, endocytosis, phagocytosis, cell migration and other processes important for cellular functions during development and in specialized cell types such as neurons, adipocytes and immune cells. My studies have identified new components of ER-PM MCS, the ER-localized extended-synaptotagmin proteins (E-Syt1, E-Syt2 and E-Syt3) whose overexpression induces formation of cortical ER while their loss of function reduces the number of ER-PM contacts. One of the important conclusions of my studies on the E-Syts is that they form a novel class of ER-PM contacts. However their functions remains to be elucidated. E-Syts have multiple lipid binding domains, including an SMP domain and multiple C2 domains. The last C2 domain of E-Syt2 and E-Syt3 bind specifically and with high affinity to PI(4,5)P2, thus bringing the ER to the PM. The SMP domain is predicted to adopt a hydrophobic lipid-binding tunnel structure, and possibly function to transfer lipids between membranes. The central objective of this proposal is to understand the functions of E-Syts at ER-PM contact sites in mammalian cells. Our working hypothesis is that regulation of PM lipid metabolism is a key function of E-Syt-mediated ER-PM contact. My preliminary results have revealed interactions between E-Syts and proteins involved in lipid metabolism, including an ER-localized phosphatidylserine synthase and two members of the oxysterol-binding protein (OSBP)-related protein (ORP) family. One of these ORPs has recently been shown to bind to phosphatidylserine rather than sterols, and likely transports this phospholipid using a PI4P exchange mechanism similar to that demonstrated for Osh4 and OSBP. These exciting preliminary results suggest a function for the E-Syts in coordinating multiple lipid synthesis and transfer activities at ER-PM MCS, opening the door to novel lipid-based regulatory mechanisms for PM membrane remodeling processes. I will explore whether the presence of these contact sites might regulate endocytosis and phagocytosis, for example by locally altering PM lipid composition. PI(4,5)P2 is required at multiple stages during clathrin-mediated endocytosis, binding to numerous endocytic adaptors and regulators. Indeed, my preliminary studies have shown that EGF receptor endocytosis is controlled by the E-Syts. I will pursue these initial studies by completing screens for interacting partners, and validating partners identified. I will also determine whether E-Syt-mediated ER-PM contacts regulate the lipid composition of the PM, and elucidate the mechanisms involved. Using my expertise in electron microscopy, live cell imaging and fluorescence microscopy, optogenetics, as well as biochemical approaches, I will study the roles of E-Syts and their lipid regulatory functions in PM remodelling processes including endocytosis and phagocytosis in macrophages.

Key physiological functions, such as cell division, migration and morphogenesis, rely on proper regulation of actin filament assembly and disassembly. While actin-binding proteins (ABPs) are well-established regulators of actin dynamics, direct post-translational modifications (PTMs) of actin are currently emerging as new crucial determinants of actin assembly. Yet, little is known about their impact on cells. Strikingly, the oxidation of specific methionine residues in actin filaments can lead to complete actin disassembly in vitro. Far from being the mere consequence of reactive oxygen species, actin oxidation is actually enzymatically controlled by mono-oxygenases of the MICAL family that specifically target actin filaments in animal cells. MICAL oxidases have recently been implicated in several cell processes and in a wide range of pathologies, including neurological disorders, susceptibility to infection, and various cancers. Conversely, actin reduction seems to be regulated by specific reductases that counteract MICAL-mediated oxidation. However, little is known regarding the function of reductases in regulating actin in cells. The newly discovered role of actin oxidation and reduction thus provides a unique window to explore how specific PTMs can reversibly and locally control the assembly of the actin cytoskeleton. Recently, the two partners of this proposal unexpectedly revealed that a MICAL family member (MICAL1) is essential for normal cytokinetic abscission, the last step of cell division leading to the physical separation of the daughter cells, by locally regulating actin depolymerization at the abscission site. The potential identity and the antagonistic role of reductases in the context of cell division are unknown. In addition, while oxidized filaments have been shown to be more sensitive to cofilin, it is unclear which of the several reactions involving cofilin, alone or in synergy with other ABPs, depend on the filament’s redox state. Also, while it is becoming clear today that the specific organization of actin filaments and their mechanical context in cells participates in the regulation of their (dis)assembly, nothing is known about the possible coupling of these factors to the filaments’ redox state. One of the key and exciting questions today is thus to understand how the interplay between actin oxidation/reduction, ABPs and the actin network architecture control actin disassembly/assembly, both at the filament level and at the cellular level. The RedoxActin project will tackle this central question, by combining state-of-the-art approaches, both at the cell scale and at the single filament level in vitro. The reductase(s) responsible for counteracting the effects of MICAL1-induced oxidation will be identified, and their contribution to the regulation of actin filament turnover will be elucidated. The coupling of the redox balance to the action of cofilin and other ABPs will be investigated, and the contribution of the filament network’s organization and applied mechanical stress will be explored, both in cells and in vitro. Together, these results are expected to open conceptually new avenues for several fields in biology, by elucidating how oxidoreductive PTMs can be balanced in cells to control actin turnover. They will also lay the ground for progress in medical research, since MICAL-induced oxidation as well as defects in cytokinesis are linked to several pathologies.

Filopodia and stereocilia are two typical membrane protrusions, that perform specific functions related to the integration of mechanical cues. Filopodia are specialized dynamic actin-rich protrusions with emerging roles in the probing of cell surroundings (ECM, neighboring cells, ...) and guidance of cell migration. An amazing feature of hair cell stereocilia, essential for their function, is the staircase morphology of the bundles which implies the precise control of the actin-filled protrusion length by the interaction of myosins motors and actin binding regulators. Current models of stereocilia growth highlight the importance of the actin-filament growth control at the tip while the rest of the stereocilia core remains stable. The marked differences in bundle size, width and actin turnover between filopodia and stereocilia are attributed to the differences in the activity of actin elongators at the tip, but also of the actin bundling proteins and myosin motors involved in building these cellular protrusions, namely Myo10 in filopodia and Myo3 in stereocilia. Though the role of myosin motors has frequently been limited to cargo tip delivery, there is now evidence that myosins are required to initiate the bundling of parallel actin filaments and play exquisite role in the control of the stereocilia bundles in concert with actin binding proteins. In particular, Myo3 forms a very intriguing ternary complex with espin-1, a protein known to bundle actin filaments. It is thus essential to focus our attention on how myosin and actin regulatory proteins work cooperatively to gain insights into the molecular events controlling filopodia and stereocilia length. We propose to investigate the similarities and differences of the Myo10 and Myo3 motors, their multiple activities on actin bundle dynamics. The originality of the Myo’N’Ease project relies on the unique combination of structural approaches with the study of actin assembly dynamics using in vitro reconstituted actin networks in a microfluidics system. Insights from structural studies will be tested in precise assays that reconstitute actin structures in physiological biochemical conditions, which is essential to propose a valuable mechanistic description.

Adhesion is a fundamental property in cell biology, regulating many processes including cell shape and differentiation. At the molecular level, adhesion is mediated by specific membrane-associated ligand-receptor pairs. Several families of adhesion molecules, including the neurexin-neuroligin complex, have been shown to play critical roles in synapse formation in neurons. Genetic mutations in these molecules are associated with neurodevelopmental diseases in humans, highlighting their importance in brain function. Despite these advances, many issues remain unclear regarding the role of adhesion in synapse organization and function. In particular, NRXs and NLGs contain multiple isoforms and splice variants, known to associate through a specific code that specifies synapse types, but how the intrinsic binding properties between the different partners govern synapse structure, dynamics, and strength is still unresolved. Our central research hypothesis is that by regulating the interaction kinetics between adhesion complexes, neurons can modulate the morphology and strength of their synaptic contacts, with implications in neuronal physiology. To explore this concept, we will manipulate synaptic complexes through the expression of a repertoire of neurexin and neuroligin isoforms/splice variants and their competitors (MDGAs, LRRTMs), and search for correlations between adhesion dynamics and synaptic parameters including structure, morphology, and functional differentiation. To answer the scientific question formulated above, we raise four specific objectives: Objective 1: To characterize the adhesion kinetics of membrane-bound synaptic complexes We will micropattern adhesion protein ectodomains and characterize their interaction kinetics with fluorescently tagged counter receptors in heterologous cells. Objective 2: To determine the effects of ligand-receptor adhesion on synapse differentiation in vitro We will design the dual micropatterning of different protein ligands to study the role of adhesion synergy and competition on synapse differentiation in cultured neurons. Objective 3: To explore the dynamics and nanoscale distribution of synaptic adhesion complexes We will monitor adhesion protein dynamics by single molecule tracking and their localization in extra-synaptic and synaptic compartments by correlative light-electron microscopy (CLEM). Objective 4: To characterize the role of adhesion molecule dynamics on synapse morphology and function ex vivo We will examine the effects of neurexin and neuroligin isoforms/splice variants on synapse morphology and physiology by 3D live imaging and electrophysiology in brain slices. To reach these objectives, we are building a highly interdisciplinary consortium of four partners: Partner 1: O. THOUMINE (DR1 CNRS, Bordeaux) will bring his knowledge of neuronal adhesion, genetic tools, cultures from transgenic mice, labelling strategies with small monomeric probes, single molecule imaging, and computer simulations. Partner 2: V. STUDER (DR2 CNRS, Bordeaux) will bring his expertise in micro-engineering and microscopy, notably 2D photopatterning and 3D structured illumination. Partner 3: ALVEOLE S.A. (Paris) will provide its latest prototypes and patterning protocols, and dedicated software development toolkits. Partner 4. J.-M. VERBAVATZ (Pr, Institut Jacques Monod, Paris) will bring his strong expertise in membrane proteins, TEM and CLEM. He is also co-director of the ImagoSeine imaging core facility (member of France BioImaging), where the EM will be performed. Overall, we expect to unravel striking correlations between adhesion dynamics and synapse development. This project will yield important new insights into relevant and timely scientific questions underlying synaptogenesis, as well as new microscopy solutions that will be commercialized by the industrial partner.