Genome-wide functional screen of lysosome biogenesis

点击翻译按钮获取中文摘要

Genome-wide screens based on RNA interference have now emerged as one powerful tool for identifying gene networks that regulate specific cellular functions of high eukaryotic cells. Lysosome biogenesis is one of the key cellular functions regulating cell homeostasis, as illustrated by more than 50 lysosomal storage disorders and related diseases in humans. Lysosome biogenesis relies on the coordinated action of several major pathways: first endocytosis, which mediates the internalization of extracellular components destined to be digested in lysosomes and, second the biosynthetic transport, which brings newly synthesized, soluble hydrolytic enzymes in order to digest endocytosed material and newly synthesized membrane proteins as part of the components constituting lysosomal membranes. Two, nonredundant pathways mediate biosynthetic transport to lysosomes: the AP-1 pathway mediates the sorting of the lysosomal enzyme receptors (the mannose 6-phosphate receptors, MPRs) and their bound-ligands whereas the AP-3 pathway mediates the sorting of the lysosomeassociated membrane glycoproteins (Lamps). In order to obtain a large, comprehensive understanding of biosynthetic transport to lysosomes and lysosome biogenesis in humans as well as its means of regulation, we wish to perform a genome-wide functional screen based on RNA interference to identify genes that regulate both the AP-1 and AP-3-dependent pathways. RNA interference is well established in the HeLa cell system used. As a read-out for performing the primary screen, we will use HeLa cells stably expressing a GFP-tagged lysosomal enzyme receptor as a marker of the AP-1-dependent pathway and follow by fluorescence microscopy changes of its steady state distribution to evaluate the genes regulating this pathway. We will also use an assay based on fluorescence to monitor changes in Lamp trafficking in order to evaluate the genes regulating the AP-3 pathway. We will first illustrate the role of ¿600 kinases and ¿100 phosphatases contained in the human genome in a RNA interference-based screen, as a first step required for establishing proofs of principal, and then achieve a screen of the entire human genome in order to establish a functional map of gene networks regulating lysosome biogenesis. During the course of our previous studies on the AP-1- and AP-3-depndent pathways, we have developed a number of quantitative assays and reagents that can be used to perform secondary screens and a further characterization of individual gene products regulating these pathways. Thus, the research described in this proposal will provide a complete functional characterization of the gene networks regulating both the AP-1- and AP-3-dependent pathways and, therefore a comprehensive functional map of core machineries and regulatory mechanisms involved in the biosynthetic transport required for lysosome biogenesis.