Identification and characterisation of genes that interact with the Drosophila MACPF protein Torso-like

The Membrane Attack Complex/Perforin-like (MACPF) protein superfamily is large and divergent, with members commonly involved in bacterial pathogenesis and vertebrate immunity. MACPF proteins are best known for their ability to oligomerise and form pores on target cell membranes. Of the -1300 MACPF proteins that have been identified so far, Torso-like (Tsl) is the only MACPF protein found in Drosophila. Instead of working in immunity, it plays a critical role in patterning of the early embryo through controlling localised activation of the receptor tyrosine kinase Torso (Tor), at the embryo termini. Tsl has been studied in this context for over twenty years, yet the mechanism by which Tsl controls localised Tor activation and whether this requires pore-formation remains unknown. Tsl is unique in the MACPF superfamily as its MACPF domain spans most of the protein, with very little flanking sequence. This is unusual as most MACPF proteins have multiple other accessory domains to carry out their functions. It therefore seems likely that Tsl interacts with partner proteins that carry such domains in order to function in terminal patterning. Furthermore, it is very likely that due to the limitations of previous genetic screens, many new terminal patterning genes are likely to have evaded detection. This thesis describes the identification of new components of the terminal patterning pathway. To identify genes acting in terminal patterning, a genome-wide modifier screen was conducted using a sensitised background induced by ectopic tsl expression. A set of 467 molecularly defined chromosomal deletions, representing 92% of the Drosophila genome, were screened. Sixty-seven genomic regions were identified that suppressed the effects of ectopic tsl expression. The causative genes in eleven suppressor regions were identified via detailed deficiency mapping and testing of available mutant alleles and RNAi lines of genes within candidate regions. Further characterisation of one of these genes, twins (tws), revealed a defect in early embryogenesis and hence it proved difficult to study its role in terminal patterning. To overcome this, tws function was studied in the larval prothoracic gland, where Tsl has a second function to regulate growth and developmental timing. tws knockdown in the prothoracic gland resulted in a developmental delay and an increased adult bodyweight. Furthermore, tws knockdown in tsl mutants showed additive interactions in both developmental timing and adult bodyweight, suggesting that tws is likely to be acting in a different pathway to tsl in this tissue. As Tsl acts independently of Tor in the prothoracic gland, tws may represent a novel regulator of the Tor pathway. Taken together, the data presented in this thesis provides a solid foundation for studying new terminal patterning genes that will further our knowledge of signalling pathway activation and MACPF function. Furthermore, there is potential for the discovery of novel Ras/MAPK signalling factors such as tws that could assist in our understanding of this highly conserved intracellular signalling cascade.