Precise control of the transition from self-renewal to terminal differentiation in stem cells is critical to maintain a balance between cell populations: an excess of stem cell self-renewal can lead to tumourigenesis, whereas an excess of differentiation can deplete the stem-cell pool. In the adult Caenorhabditis elegans germline, Notch signals emanate from the somatic distal tip cell to maintain germline stem cells (GSCs) in a proliferative state by repressing the translation of meiotic promoting factors. We have uncovered a novel pathway regulating the decision between GSC renewal and meiotic differentiation that involves the ubiquitin-proteolytic system (UPS). Using a novel temperature-sensitive allele of the cul-2 gene, we found that the CUL-2 RING E3 ubiquitin ligase, in combination with the Leucine Rich Repeat 1 substrate recognition subunit (CRL2LRR-1), negatively regulates the transition from the mitotic zone of the germline to the meiotic programme of chromosome pairing, synapsis, and recombination. More specifically, we find that CRL2LRR-1 regulates in stem cells the stability of the HORMA domain-containing protein HTP-3, which is required for loading structural proteins onto meiotic chromosomes and for the formation of the double-strand breaks that initiate meiotic recombination. Furthermore, we found that cyclin E/Cdk2 kinase, which is specifically activated in GSCs but repressed upon meiotic differentiation, phosphorylates HTP-3 and regulates its stability. Besides HTP-3, CUL-2 targets other factors for degradation to prevent precocious meiotic entry and to promote germline stem cell proliferation. Herein, we propose to use a unique combination of genetics, cell biology, biochemical and quantitative proteomic approaches to elucidate the role of protein degradation in germ cell biology. In particular, we propose to identify CUL-2 targets in the germline and the molecular mechanisms controlling their degradation in space. The role of the UPS and CUL-2 in germline stem cell biology has not been studied so far. CUL-2 is evolutionarily conserved in metazoans and appears to regulate germ cell divisions in Drosophila. Therefore emerging paradigms provided by the study of germ cell biology in C. elegans should be directly applicable in other systems and possibly also in humans.

The crucial role of the pollination activity of domestic honeybees is at risk because of an alarming increase in numbers of colony losses in the last years. Among the multiple factors that impact colony fitness, the sensitivity of honeybees to sublethal doses of insecticides, affecting crucial functions involved in foraging, including sensory processing, learning, memory and motor functions have been revealed. Acute toxicity of pyrethroids, the most commonly used insecticides, mainly occurs via binding to voltage-gated sodium (Na+) channels. However, it is clear now that calcium (Ca2+) channels are affected at similar doses. Because Na+ and Ca2+ channels are key actors of neuronal and muscular excitability, they are involved in many neural processes such as locomotion, sensory processing, learning and memory formation in vertebrates and invertebrates. They are thus both suspected of participating in sublethal toxicity pyrethroids. Oddly, the biophysical properties, regulation and pharmacology of Na+ and Ca2+ channels are still poorly studied in honeybees. Moreover, their subunit composition and molecular identity in the tissues potentially affected by these insecticides (muscle, central and peripheral nervous system) is largely unknown. We have identified in the genomic honeybee database the pore-forming subunit genes of the voltage–gated Na+ channels (2 genes? and Ca2+ channels (3 genes) as well as respectively 5 and 4 genes for regulatory subunits. Our preliminary cloning (for all Na+ and Ca2+ channels subunits) and expression studies (for one regulatory subunit) have revealed unique amino-acids sequences and specific biophysical and regulatory properties in honeybees. In this integrative project, we propose to analyse the role of these genes in honeybee physiology and behaviour (locomotion, olfactory perception, orientation, learning, memory) and in the sensitivity of this insect to sublethal doses of pyrethroids. • Task 1 will characterize the effects of sublethal doses of type I and type II pyrethroids on honeybee physiology. We will determine sublethal doses of pyrethroids on honeybees. We will then characterize their effects on Na+ and Ca2+ currents in honey bee muscle cells and central and peripheral neurons in vitro, as well as their effects and dependence on calcium homeostasis. We will also analyze the effect of pyrethroids on gene expression. • Task 2 will address the molecular characterization of Na+ and Ca2+ channel subunits identified in honeybees. We will define their expression profiles, biophysical properties and sensitivity to pyrethroids using heterologous expression. Their roles in Na+ and Ca2+ influxes in muscle cells and neurons will be challenged using siRNA and specific antibodies. Cell lines expressing actual combinations of subunits found in honeybees will be produced as screening tools for in vitro toxicological tests. • Task 3 will unravel the role of Na+ and Ca2+ channel subunits in sensory, cognitive and behavioural tasks, so that the sublethal toxicity of pyrethroids can be understood. The impact of siRNA and of sublethal doses of pyrethroids will be evaluated on olfactory processing, learning and memory, locomotor activity and free-flying foraging behaviour. This integrative approach brings together 4 partners with combined expertise in apidology, behavioural neurophysiology, toxicology, molecular cloning, electrophysiology and heterologous expression. It will improve our understanding of the neurobiology of sensory and cognitive functions in honeybees and clarify acute and chronic toxicity of pyrethroids and their effects on these processes. This project will also provide screening tools for evaluating the toxicity of phytopharmaceutical products toward these key channels.