Many metazoan cells can take up exogenous double-stranded (ds) RNA and use it to initiate an RNA silencing response, however, the mechanism for this uptake is ill-defined. Here, we identify the pathway for dsRNA uptake in Drosophila melanogaster S2 cells. Biochemical and cell biological analyses, and a genome-wide screen for components of the dsRNA-uptake machinery, indicated that dsRNA is taken up by an active process involving receptor-mediated endocytosis. Pharmacological inhibition of endocytic pathways disrupted exogenous dsRNA entry and the induction of gene silencing. This dsRNA uptake mechanism seems to be evolutionarily conserved, as knockdown of orthologues in Caenorhabditis elegans inactivated the RNA interference response in worms. Thus, this entry pathway is required for systemic RNA silencing in whole organisms. In Drosophila cells, pharmacological evidence suggests that dsRNA entry is mediated by pattern-recognition receptors. The possible role of these receptors in dsRNA entry may link RNA interference (RNAi) silencing to other innate immune responses.RNAi is a highly conserved dsRNA-guided mechanism that mediates sequence-specific gene silencing 1 . A number of animal cells can naturally take up exogenous dsRNA and use it to initiate RNAi silencing 2,3 . In some organisms, such as Drosophila, certain cells can efficiently take up dsRNA but seem to be unable to transmit this dsRNA to other cells in the body 4 . dsRNA uptake without further transmission to other cells has also been reported for some mammalian cell types [5][6][7] . Other organisms (such as C. elegans or juvenile grasshoppers) can both take up dsRNA and spread it systemically to elicit an RNAi response throughout the entire animal 8,9 . The mechanisms of uptake and spread of dsRNA are poorly understood. It is unclear whether dsRNA enters cells through passive, non-specific mechanisms, or whether there is an active mechanism that controls entry. Genetic analysis to identify genes involved in systemic spread of dsRNA in C. elegans isolated several mutants unable to distribute an ingested dsRNA signal from the gut throughout the body 8,10,11 . One of these, SID-1 (also known as RSD-8) is a putative transmembrane protein required for systemic spread 8 . When expressed ectopically in Drosophila cells, SID-1 enhanced the RNAi response observed at low dsRNA 5Correspondence should be addressed to R.A. (e-mail: Raul.Andino@UCSF.edu). 3 Current address: Canada Research Chair in Innate Immunity, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Canada 4 These authors contributed equally to this work.Note: Supplementary Information is available on the Nature Cell Biology website. COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. concentrations 12 , raising the possibility that SID-1 may function as a channel on the cell surface for uptake of dsRNA. However, endogenous sid-1 homologues have not been found in the Drosophila genome, yet Drosophila S2 cells...

Much of our understanding of animal cell cytokinesis centers on the regulation of the equatorial acto-myosin contractile ring that drives the rapid ingression of a deep cleavage furrow. However, the central part of the mitotic spindle collapses to a dense structure that impedes the furrow and keeps the daughter cells connected via an intercellular bridge. Factors involved in the formation, maintenance, and resolution of this bridge are largely unknown. Using a library of 7,216 double-stranded RNAs (dsRNAs) representing the conserved genes of Drosophila, we performed an RNA interference (RNAi) screen for cytokinesis genes in Schneider's S2 cells. We identified both familiar and novel genes whose inactivation induced a multi-nucleate phenotype. Using live video microscopy, we show that three genes: anillin, citron-kinase (CG10522), and soluble N-ethylmaleimide sensitive factor (NSF) attachment protein (alpha-SNAP), are essential for the terminal (post-furrowing) events of cytokinesis. anillin RNAi caused gradual disruption of the intercellular bridge after furrowing; citron-kinase RNAi destabilized the bridge at a later stage; alpha-SNAP RNAi caused sister cells to fuse many hours later and by a different mechanism. We have shown that the stability of the intercellular bridge is essential for successful cytokinesis and have defined genes contributing to this stability.

Phagocytosis is a highly conserved aspect of innate immunity. We used Drosophila melanogaster S2 cells as a model system to study the phagocytosis of Candida albicans, the major fungal pathogen of humans, by screening an RNAi library representing 7,216 fly genes conserved among metazoans. After rescreening the initial genes identified and eliminating certain classes of housekeeping genes, we identified 184 genes required for efficient phagocytosis of C. albicans. Diverse biological processes are represented, with actin cytoskeleton regulation, vesicle transport, signaling, and transcriptional regulation being prominent. Secondary screens using Escherichia coli and latex beads revealed several genes specific for C. albicans phagocytosis. Characterization of one of those gene products, Macroglobulin complement related (Mcr), shows that it is secreted, that it binds specifically to the surface of C. albicans, and that it promotes its subsequent phagocytosis. Mcr is closely related to the four Drosophila thioester proteins (Teps), and we show that TepII is required for efficient phagocytosis of E. coli (but not C. albicans or Staphylococcus aureus) and that TepIII is required for the efficient phagocytosis of S. aureus (but not C. albicans or E. coli). Thus, this family of fly proteins distinguishes different pathogens for subsequent phagocytosis.

Studies in mammals uncovered important signaling roles of nitric oxide (NO), and contributions to innate immunity. Suggestions of conservation led us to explore the involvement of NO in Drosophila innate immunity. Inhibition of nitric oxide synthase (NOS) increased larval sensitivity to gram-negative bacterial infection, and abrogated induction of the antimicrobial peptide Diptericin. NOS was up-regulated after infection. Antimicrobial peptide reporters revealed that NO triggered an immune response in uninfected larvae. NO induction of Diptericin reporters in the fat body required immune deficiency (imd) and domino. These findings show that NOS activity is required for a robust innate immune response to gram-negative bacteria, NOS is induced by infection, and NO is sufficient to trigger response in the absence of infection. We propose that NO mediates an early step of the signal transduction pathway, inducing the innate immune response upon natural infection with gram-negative bacteria.