Genetic proteomic and functional analysis of junctional complexes in Drosophila

Lead Research Organisation: University of Glasgow
Department Name: School of Life Sciences

Abstract

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.

Technical Summary

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.
 
Description Like us, insects regulate their internal environment by pumping in desirable compounds, and excreting waste products, across the sheets of cells, or epithelia (like gut, lung, kidney in us) that separate their different body compartments.
Our lab has a long published history in trying to establish how insects accomplish this job. This is because some of the remarkable success of this Class of life - there are more species of insects than all other living things combined - can be ascribed to their ability to regulate their internal environments in a range of harsh conditions.
In this award, we studied the junctions between epithelial cells that isolate different microenvironments from each other. Sheets of cells are neither strong, nor impermeable, unless they are 'zipped together' with tight junctions. Disorders of junctional function can be disastrous - for example, MS in humans results from a leaky blood-brain barrier, allowing the immune system to 'discover' and attack the CNS. Given the success of insects, we hoped that understanding their unique junctions (known as septate junctions), we might unravel new, selective targets for insect control. Additionally, our target of interest, the Malpighian (kidney) tubule, can move fluid faster than any other epithelium known, on a per-cell basis. This has led to the suggestion that ions and water may have to move paracellullarly (through the junctions between cells), rather than transcellularly (through transporters and channels in the cells themselves) to achieve such high flow rates.
We have been able to show that junctions are essential for the survival of the insect, and for normal functioning of the kidney epithelium; additionally, we have been able to confirm that chloride moves transcellularly through channels, not paracellularly through the junctions. This finding resolved a long-standing article in the field, and was published in PNAS.
Exploitation Route Our data suggest that certain junctional proteins are essential for normal development and function of epithelia in insects. Additionally, we have identified a single chloride channel (ClC-a) as the key player in allowing chloride to cross the insect 'kidney' tubule. Such proteins are potentially targets for new, more selective insecticides.
Sectors Agriculture, Food and Drink