GENETIC SCREENING TO IDENTIFY INTERACTORS OF ESCRT-II SUBUNIT, VPS25, AND PRELIMINARY CHARACTERISATION OF CANDIDATES

ESCRT (Endosomal Sorting Complex Required for Transport) proteins regulate cell surface receptor degradation by sorting and packaging ubiquitinated cargoes into the intraluminal vesicles of multivesicular bodies (MVBs). A range of human diseases including cancer, and neurodegeneration display altered expression or are caused by mutations of ESCRT subunits. Studies have shown that Drosophila tissues lacking ESCRTs display neoplastic-like features like overproliferation and polarity defects, partly due to aberrant signalling including Notch signalling. To understand ESCRT-regulated processes in vivo, we utilised modification of a deformed wing phenotype specifically caused by knockdown (RNAi) of Vps25, an ESCRT-II subunit. We systematically screened chromosomal regions and identified 204 genetic interactors of Vps25 that enhanced/suppressed the phenotype. They include genes that function in trafficking, signalling, transcription, ion transport and many other biological processes; suggesting that ESCRTs influence a wide range of biological processes. We have focused on a subset of these hits that regulate tissue growth with a secondary screen based on modification of a Delta-driven eye overgrowth phenotype, isolating a subset of 43 genes involved in regulating tissue growth, some of which are novel and uncharacterised. Human orthologues of some of these genes are important in cancers; dropout (dop), whose mammalian orthologues are the MAST kinases, have been shown to contribute towards breast cancer development. dop mediates Delta-driven eye overgrowth possibly by upregulating Delta expression. In human cells, MAST2 does not affect Notch signalling but might contribute to tumorigenesis by regulating the NFκB pathway. We have also characterised another interactor, CG12163 which is the homologue of mammalian Cathepsin F. Mutations in Cathepsin F cause a rare form of neuronal ceroid lipofuscinosis (NCL) called Type B Kufs disease. Our Drosophila model which recapitulates aspects of the human disease phenotype suggests that defects in autophagy might underlie the pathogenesis of NCLs.