Damage initiates a pleiotropic cellular response aimed at cellular survival when appropriate. To identify genes required for damage survival, we used a cell-based RNAi screen against the Drosophila genome and the alkylating agent methyl methanesulphonate (MMS). Similar studies performed in other model organisms report that damage response may involve pleiotropic cellular processes other than the central DNA repair components, yet an intuitive systems level view of the cellular components required for damage survival, their interrelationship, and contextual importance has been lacking. Further, by comparing data from different model organisms, identification of conserved and presumably core survival components should be forthcoming. We identified 307 genes, representing 13 signaling, metabolic, or enzymatic pathways, affecting cellular survival of MMS-induced damage. As expected, the majority of these pathways are involved in DNA repair; however, several pathways with more diverse biological functions were also identified, including the TOR pathway, transcription, translation, proteasome, glutathione synthesis, ATP synthesis, and Notch signaling, and these were equally important in damage survival. Comparison with genomic screen data from Saccharomyces cerevisiae revealed no overlap enrichment of individual genes between the species, but a conservation of the pathways. To demonstrate the functional conservation of pathways, five were tested in Drosophila and mouse cells, with each pathway responding to alkylation damage in both species. Using the protein interactome, a significant level of connectivity was observed between Drosophila MMS survival proteins, suggesting a higher order relationship. This connectivity was dramatically improved by incorporating the components of the 13 identified pathways within the network. Grouping proteins into "pathway nodes" qualitatively improved the interactome organization, revealing a highly organized "MMS survival network." We conclude that identification of pathways can facilitate comparative biology analysis when direct gene/orthologue comparisons fail. A biologically intuitive, highly interconnected MMS survival network was revealed after we incorporated pathway data in our interactome analysis.
Lipid droplets are ubiquitous triglyceride and sterol ester storage organelles required for energy storage homeostasis and biosynthesis. Although little is known about lipid droplet formation and regulation, it is clear that members of the PAT (perilipin, adipocyte differentiation related protein, tail interacting protein of 47 kDa) protein family coat the droplet surface and mediate interactions with lipases that remobilize the stored lipids. We identified key Drosophila candidate genes for lipid droplet regulation by RNA interference (RNAi) screening with an image segmentation-based optical read-out system, and show that these regulatory functions are conserved in the mouse. Those include the vesicle-mediated Coat Protein Complex I (COPI) transport complex, which is required for limiting lipid storage. We found that COPI components regulate the PAT protein composition at the lipid droplet surface, and promote the association of adipocyte triglyceride lipase (ATGL) with the lipid droplet surface to mediate lipolysis. Two compounds known to inhibit COPI function, Exo1 and Brefeldin A, phenocopy COPI knockdowns. Furthermore, RNAi inhibition of ATGL and simultaneous drug treatment indicate that COPI and ATGL function in the same pathway. These data indicate that the COPI complex is an evolutionarily conserved regulator of lipid homeostasis, and highlight an interaction between vesicle transport systems and lipid droplets.
Nearly 1.7 billion people are infected with Mycobacterium tuberculosis. Its ability to survive intracellularly is thought to be central to its success as a pathogen, but how it does this is poorly understood. Using a Drosophila model of infection, we identify three host cell activities, Rab7, CG8743, and the ESCRT machinery, that modulate the mycobacterial phagosome. In the absence of these factors the cell no longer restricts growth of the non-pathogen Mycobacterium smegmatis. Hence, we identify factors that represent unique vulnerabilities of the host cell, because manipulation of any one of them alone is sufficient to allow a nonpathogenic mycobacterial species to proliferate. Furthermore, we demonstrate that, in mammalian cells, the ESCRT machinery plays a conserved role in restricting bacterial growth.
RNA interference has re-energized the field of functional genomics by enabling genome-scale loss-of-function screens in cultured cells. Looking back on the lessons that have been learned from the first wave of technology developments and applications in this exciting field, we provide both a user's guide for newcomers to the field and a detailed examination of some more complex issues, particularly concerning optimization and quality control, for more advanced users. From a discussion of cell lines, screening paradigms, reagent types and read-out methodologies, we explore in particular the complexities of designing optimal controls and normalization strategies for these challenging but extremely powerful studies.