Genetic proteomic and functional analysis of junctional complexes in Drosophila

Our bodies rely on the integrity of our barrier epithelia; the sheets of cells (like skin, gut, lung) that separate us from the outside world, or keep different regions of our bodies apart. The thousands of cells in such epithelia have highly specialised junctions that zip them together, in order to prevent leakage (a 'tight' epithelium) or to permit flux of only specific solutes (a 'leaky' epithelium). Problems with this integrity can be serious or fatal (e.g. peritonitis, multiple sclerosis, kidney disease). What makes a junction either tight or leaky? Many of the proteins that are found in junctions are already known. However, with the exceptions of some human genetic diseases and a few 'knockout' mouse lines, it is hard to tease apart the contributions of each protein. We propose to study the septate junction (the homologue of the vertebrate 'tight' junction) in the kidney of the model insect, Drosophila melanogaster. This is an ideal system, because we can cheaply and quickly intervene in gene function in cells of our choice. We will use proteomics to identify the major proteins in the junction, then mutate the genes implicated by the proteomics results, and study whether the junctions remain tight, or become leaky. This will allow a comprehensive study of the relative roles of the proteins that make up the junction.

Tight junctions, or septate junctions (their insect anologues) are found in tissues where relative impermeability is considered necessary. Much is known about tight junctional function; however, the roles of many of the junctional proteins remain obscure. because of the difficulty and cost of producing multiple transgenic lines. Additionally, it is not clear just how important this impermeability is: if a junction is made more leaky experimentally, does it make a big difference to epithelial function - or to organismal viability? Many of the genes underlying septate junction formation are already known in Drosophila through genetic analysis of development. Transgenic intervention in gene function is remarkably potent and specific; it is possible to modulate the function of single genes in specific cell types in an otherwise normal insect. However, physiological analysis is famously hard in this tiny model. We propose a multi-stranded approach to the smooth septate junction in Drosophila, using the renal (Malpighian) tubule, which provides a remarkably informative and quantitative phenotype for transport and cell signalling genes. We will identify known junctional genes that are expressed in tubule from our online atlas of gene expression (flyatlas.org), and obtain or generate mutants and epitope-tagged transgenic lines. We will use these, and using antibodies that we have already shown to label junctions in the tubule, to identify novel proteins associated with the lateral membrane of the tubule by pull-downs, and by epitope-tagging known septate junctional proteins. Genes implicated in junctional function will be mutagenised (over-expression cf. RNAi alleles, fluorescently tagged proteins, etc), where useful alleles do not already exist, and the resulting impact on both insect survival and renal function analysed. This will provide perhaps the most integrated genetic, proteomic and functional analysis of such junctions to date.