Malaria parasites critically depend on an unusual form of gliding motility to colonize their hosts and to invade cells. Transmission of malaria to the mosquito, for instance, relies on the ability of the motile zygote, the ookinete, to glide towards and invade the midgut epithelium. Results obtained in the host team have revealed a central role of cGMP- and calcium-dependent signalling in ookinete motility. Nevertheless, how these pathways control gliding is unknown. I therefore propose to combine real-time quantitative imaging approaches with molecular phenotyping of parasite phosphoproteins to identify and distinguish molecular targets of signalling through calcium and cGMP-dependent pathways in Plasmodium berghei ookinetes. For this purpose, I will use transgenic parasites in which cGMP- and/or calcium-dependent signalling pathways are either inactivated and/or deregulated. The impact of genetically inhibiting or over-stimulating specific protein kinases on motility will be analysed in an automated quantitative imaging assay, allowing signalling pathways to be linked with specific changes in motility phenotypes. I will then use shotgun phosphoproteomics on enriched populations of selected mutants to identify candidate substrates. Phenotyping of stage-specific mutants in candidate substrates will allow the molecular targets of signalling cascades to be verified.