The size of genomes compared to cell dimensions imposes a necessity of DNA condensation. Although DNA is highly condensed, it should nonetheless allow gene expression and accurate segregation of genetic material to daughter cells at each cell division. We have shown that condensation of the chromosome in the bacterial model, Escherichia coli, relies on the existence of large insulated regions called Macrodomains (MD) that coexist with 2 regions called Non-Structured Regions. We have recently identified one factor specifying the properties of one MD; thus we will be able to elucidate the molecular mechanisms responsible for the condensation and insulation in the cell of large chromosomal regions. Characterization of the processes governing the structuring of other MDs should enable us to determine whether other strategies are operant in E. coli cells. Affecting macrodomain formation will enable us to highlight the rationale of chromosome organization into macrodomains and Non-structured regions and characterize chromosome management during the cell cycle.

La conversion de l'énergie chimique en énergie mécanique par les kinésines permet aux eucaryotes de modifier l'organisation de leur contenu cellulaire. Pour ce, les rôles des kinésines et des microtubules sont étroitement liés puisque ces derniers sont les 'rails' sur lesquels les kinésines se déplacent. L'activité biochimique des kinésines est bien caractérisée et leur repliement est connu. Par contre, l'interaction de ces protéines avec les microtubules et les changements de cette interaction qui leur permettent d'utiliser l'énergie issue de l'hydrolyse de l'ATP restent largement incompris. Notre objectif est de déterminer la structure de complexes de la tubuline avec des kinésines, de façon à définir à l'échelle atomique les mécanismes de fonctionnement de ces protéines. Ceci devrait apporter des informations sur les deux processus cellulaires fondamentaux, le traffic cellulaire dépendant des microtubules et la dépolymérisation du cytosquelette microtubulaire. L'obstacle majeur est l'obtention d'un complexe kinésine-tubuline homogène et monodisperse qui permette d'entreprendre des essais de cristallisation dans de bonnes conditions. La plupart des kinésines ont une forte affinité pour les microtubules mais une très faible affinité pour la tubuline soluble ; leurs complexes avec la tubuline ne se prêtent donc pas à une analyse structurale à haute résolution. Nous mettrons à profit la forte affinité entre la tubuline soluble et les kinésines dont le domaine moteur est en position interne de la séquence pour isoler un complexe tubuline kinésine qui se prête à une analyse radiocristallographique. La caractérisation par radiocristallographie de ces complexes nécessitera deux développements : - la production de fragments monomériques de kinésines à domaine moteur interne solubles, fonctionnels et ayant une forte affinité pour la tubuline ; - la formation de complexes monodisperses, en évitant en particulier la propension de la tubuline à former des agrégats hélicoïdaux et des anneaux de tailles variables. Pour ce qui concerne le premier aspect, nous utiliserons des approches classiques de génétique moléculaire, appliquées aux kinésines à domaine moteur interne identifiées chez l'homme, la souris et chez Plasmodium falciparum. Pour ce qui concerne le deuxième aspect, nous testerons l'utilité de deux types de complexes qui empêchent l'agrégation de la tubuline : - les complexes avec des domaines de type stathmine ou leurs fragments qui suffisent à séquestrer la tubuline - les complexes avec des dérivés de la vinblastine conçus de façon à empêcher la fixation de l'un des deux hétérodimères de tubuline qui se fixent normalement à cette petite molécule. La structure de la tubuline soluble étant connue ainsi que le repliement des kinésines, nous utiliserons, dans un premier temps, le remplacement moléculaire pour déterminer la structure du complexe.

The cerebral cortex is considered as the most integrative structure of the mammalian brain. It processes inputs from multiple brain areas through a complex neuronal network and is involved in many cognitive functions. This structure therefore constitutes a central subject of research in modern Neuroscience which aims to understand the neuronal bases of behaviour. Because of its high degree of organization, the rodent’s whisker-to-barrel cortex pathway is a model of choice to study the cortical processing of sensory information. The neuronal actors and the cascade of excitatory synaptic events that underlies this sensory pathway have been largely studied using classical anatomical and electrophysiological techniques from in vitro brain slices preparations or in vivo, in anesthetised animals. However, little is known about the neural mechanisms underlying sensory processing and perception in awake behaving animals. Recent progresses in the technology related to voltage sensitive dye imaging has opened new avenues in this field, allowing the recording of cortical ensemble activity with a temporal resolution reaching the millisecond and a spatial resolution of few tens of micrometers in the awake mouse sensorimotor cortex. Such recordings have revealed a strong influence of the behaviour on the spatiotemporal properties of the cortical responses induced by tactile stimulations. At the neuronal network level, the basalo-cortical cholinergic fibers, which project broadly to the cerebral cortex and whose impairment affect motivational and arousal states, are good candidates to be involved in this behavioural modulation of the cortical sensory processing. Because they can exert a rapid excitation of specific cortical interneuron populations via ligand-gated ion channels, they could notably contribute to shape the cortical activation induced by tactile stimulation. In order to study the impact of cholinergic inputs on cortical sensory processing we will focus on the model system of the mouse whisker-to-barrel cortex sensory pathway. The modulation of cortical responses evoked by tactile stimulation will be studied by in vivo voltage sensitive dye imaging and multiple single-unit recordings combined with the activation or inhibition of cholinergic release from basalo-cortical fibers using optogenetic tools. This novel experimental approach combines genetic and optical methods to achieve either activation or silencing of chosen neuronal populations with high spatial and temporal precision. It is based on the genetically targeted selective expression of microbial light-activated proteins (i.e. light-gated cation channel for neuronal excitation, or light drivable proton pump for neuronal silencing). By using Cre-driver mice combined with recombinase-dependent viral vectors, we will drive specifically the expression of such light-activated proteins in basalo-cortical cholinergic neurons, and therefore get the ability to control precisely and specifically their activity. The realisation of this project will certainly help us to describe the implication of ascending cholinergic neuromodulatory projections on cortical function, and to further the understanding of the mechanisms underlying the cognitive deficits associated with their degeneration.

The project proposes a comprehensive study of the evolution of male olfactory processing structures dedicated to sexual communication in honeybees of the genus Apis, at the species and at the subspecies level. To this goal, it will use a range of techniques including neuroanatomy, neurophysiology, molecular biology and behaviour. Honeybees constitute a prominent scientific model and a worldwide crucial agricultural agent for pollination and honey production. Although they have been intensively studied, central aspects of their mating behaviour and physiology are utterly unknown. Honeybees display striking mating behaviour, males (drones) gathering high in the air at discrete congregation areas, where they engage in scramble competition to mate with virgin queens, at which point they die. Attraction to the queen is mainly mediated by olfactory cues. Queens produce a wealth of pheromones, best studied for their role on worker physiology and behaviour, thereby maintaining the cohesion of the hive. Of these, one component, 9-ODA, was shown to attract drones. However, the existence of co-attractants is unknown. We know nevertheless that the brain of the drone harbours in its primary olfactory centre, the antennal lobe, four pheromone-processing units, the macroglomeruli (MG). Previous work carried out by the Project Coordinator using in vivo calcium imaging demonstrated that the largest MG actually processes 9-ODA information, but the role of the remaining units is unknown. Aim 1 of this project is to unravel the pheromonal communication system of the drone in the standard species, the western honeybee Apis mellifera. The genus Apis comprises 9 sympatric and allopatric species found mostly in south-east Asia. They can be classified in three main phylogenetic groups: the dwarf honeybees (2 species), the giant honeybees (2 species) and the cavity-nesting honeybees (5 species including Apis mellifera). Due to their more simple organization and cognitive abilities, the dwarf species are thought to have diverged first and the cavity-nesting bees would represent the more derived species in the bee phylogeny. Previous work has described colony organization, task allocation and foraging behaviour among others in these species. However, very little is known about the evolution of sexual communication in honeybee, although the main attractant (9-ODA) seems to be conserved in most – but not all species. Moreover, the existence of co-attractants is unknown. Cross-species comparisons of the pheromonal components suggest that the more derived cavity-nesting species present a higher number of mandibular queen pheromone components. This enrichment of the queen pheromone through evolution, could find its parallel in an apparent complexification of the pheromone-specific processing units in the drone antennal lobe. Aim 2 of this project asks how sexual pheromonal communication evolved in bees of the Apis genus. Was the observed improvement in social organization and cognitive abilities accompanied by a higher elaboration of sexual communication? Variations in sexual pheromonal communication may also take place between subspecies and play a role in ongoing speciation phenomena. The western honeybee A. mellifera is distributed on a wide geographical range, with 24 different subspecies living in very different climates from cold temperate to tropical, from permanent humidity to semi-desert. The subspecies show strong behavioural differences concerning colony defence, swarming, orientation, recruitment and foraging behaviour, especially flower constancy. By contrast, little is known about possible adaptations of their mating behaviour, and no study has studied the specifics of olfactory sexual communication and odour representation in different subspecies. Aim 3 of this project will attempt to identify race-specific modifications of the anatomy and function of the pheromone-specific units of the drone antennal lobe.