Understanding human gene function is fundamental to understanding and treating diseases. Research using the model organism Drosophila melanogaster benefits from a wealth of molecular genetic resources and information useful for efficient in vivo experimentation. Moreover, Drosophila offers a balance as a relatively simple organism that nonetheless exhibits complex multicellular activities. Recent examples demonstrate the power and continued promise of Drosophila research to further our understanding of conserved gene functions.
Phagocytosis is important during development and in the immune response for the removal of apoptotic cells and pathogens, yet its molecular mechanisms are poorly understood. In Caenorhabditis elegans, the CED2/5/10/12 pathway regulates actin during phagocytosis of apoptotic cells, whereas the role of the CED1/6/7 pathway in phagocytosis is unclear. We report that Undertaker (UTA), a Drosophila Junctophilin protein, is required for Draper (CED-1 homolog)-mediated phagocytosis. Junctophilins couple Ca2+ channels at the plasma membrane to those of the endoplasmic reticulum (ER), the Ryanodine receptors. We place Draper, its adaptor drCed-6, UTA, the Ryanodine receptor Rya-r44F, the ER Ca2+ sensor dSTIM, and the Ca2+-release-activated Ca2+ channel dOrai in the same pathway that promotes calcium homeostasis and phagocytosis. Thus, our results implicate a Junctophilin in phagocytosis and link Draper-mediated phagocytosis to Ca2+ homeostasis, highlighting a previously uncharacterized role for the CED1/6/7 pathway.
Most studies of host-pathogen interactions have focused on pathogen-specific virulence determinants. Here, we report a genome-wide RNA interference screen to identify host factors required for intracellular bacterial pathogenesis. Using Drosophila cells and the cytosolic pathogen Listeria monocytogenes, we identified 305 double-stranded RNAs targeting a wide range of cellular functions that altered L. monocytogenes infection. Comparison to a similar screen with Mycobacterium fortuitum, a vacuolar pathogen, identified host factors that may play a general role in intracellular pathogenesis and factors that specifically affect access to the cytosol by L. monocytogenes.
Genetic screens in the yeast Saccharomyces cerevisiae have identified many proteins involved in the secretory pathway, most of which have orthologues in higher eukaryotes. To investigate whether there are additional proteins that are required for secretion in metazoans but are absent from yeast, we used genome-wide RNA interference (RNAi) to look for genes required for secretion of recombinant luciferase from Drosophila S2 cells. This identified two novel components of the secretory pathway that are conserved from humans to plants. Gryzun is distantly related to, but distinct from, the Trs130 subunit of the TRAPP complex but is absent from S. cerevisiae. RNAi of human Gryzun (C4orf41) blocks Golgi exit. Kish is a small membrane protein with a previously uncharacterised orthologue in yeast. The screen also identified Drosophila orthologues of almost 60% of the yeast genes essential for secretion. Given this coverage, the small number of novel components suggests that contrary to previous indications the number of essential core components of the secretory pathway is not much greater in metazoans than in yeasts.
Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca(2+) influx and Ca(2+) release-activated Ca(2+) (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca(2+) influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca(2+) entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca(2+) pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca(2+) depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.
Wnt proteins are secreted, lipid-modified glycoproteins that control animal development and adult tissue homeostasis. Secretion of Wnt proteins is at least partly regulated by a dedicated machinery. Here, we report a genome-wide RNA interference screen for genes involved in the secretion of Wingless (Wg), a Drosophila Wnt. We identify three new genes required for Wg secretion. Of these, Emp24 and Eclair are required for proper export of Wg from the endoplasmic reticulum (ER). We propose that Emp24 and Eca act as specific cargo receptors for Wg to concentrate it in forming vesicles at sites of ER export.
Chlamydia spp. are intracellular obligate bacterial pathogens that infect a wide range of host cells. Here, we show that C. caviae enters, replicates, and performs a complete developmental cycle in Drosophila SL2 cells. Using this model system, we have performed a genome-wide RNA interference screen and identified 54 factors that, when depleted, inhibit C. caviae infection. By testing the effect of each candidate's knock down on L. monocytogenes infection, we have identified 31 candidates presumably specific of C. caviae infection. We found factors expected to have an effect on Chlamydia infection, such as heparansulfate glycosaminoglycans and actin and microtubule remodeling factors. We also identified factors that were not previously described as involved in Chlamydia infection. For instance, we identified members of the Tim-Tom complex, a multiprotein complex involved in the recognition and import of nuclear-encoded proteins to the mitochondria, as required for C. caviae infection of Drosophila cells. Finally, we confirmed that depletion of either Tom40 or Tom22 also reduced C. caviae infection in mammalian cells. However, C. trachomatis infection was not affected, suggesting that the mechanism involved is C. caviae specific.
Regulation of cell growth is a fundamental process in development and disease that integrates a vast array of extra- and intracellular information. A central player in this process is RNA polymerase I (Pol I), which transcribes ribosomal RNA (rRNA) genes in the nucleolus. Rapidly growing cancer cells are characterized by increased Pol I-mediated transcription and, consequently, nucleolar hypertrophy. To map the genetic network underlying the regulation of nucleolar size and of Pol I-mediated transcription, we performed comparative, genome-wide loss-of-function analyses of nucleolar size in Saccharomyces cerevisiae and Drosophila melanogaster coupled with mass spectrometry-based analyses of the ribosomal DNA (rDNA) promoter. With this approach, we identified a set of conserved and nonconserved molecular complexes that control nucleolar size. Furthermore, we characterized a direct role of the histone information regulator (HIR) complex in repressing rRNA transcription in yeast. Our study provides a full-genome, cross-species analysis of a nuclear subcompartment and shows that this approach can identify conserved molecular modules.
Few components of the mitochondrial fission machinery are known, even though mitochondrial fission is a complex process of vital importance for cell growth and survival. Here, we describe a novel protein that controls mitochondrial fission. This protein was identified in a small interfering RNA (siRNA) screen using Drosophila cells. The human homologue of this protein was named Mitochondrial fission factor (Mff). Mitochondria of cells transfected with Mff siRNA form a closed network similar to the mitochondrial networks formed when cells are transfected with siRNA for two established fission proteins, Drp1 and Fis1. Like Drp1 and Fis1 siRNA, Mff siRNA also inhibits fission induced by loss of mitochondrial membrane potential, it delays cytochrome c release from mitochondria and further progression of apoptosis, and it inhibits peroxisomal fission. Mff and Fis1 are both tail anchored in the mitochondrial outer membrane, but other parts of these proteins are very different and they exist in separate 200-kDa complexes, suggesting that they play different roles in the fission process. We conclude that Mff is a novel component of a conserved membrane fission pathway used for constitutive and induced fission of mitochondria and peroxisomes.
Mitochondria are integral components of cellular calcium (Ca2+) signaling. Calcium stimulates mitochondrial adenosine 5'-triphosphate production, but can also initiate apoptosis. In turn, cytoplasmic Ca2+ concentrations are regulated by mitochondria. Although several transporter and ion-channel mechanisms have been measured in mitochondria, the molecules that govern Ca2+ movement across the inner mitochondrial membrane are unknown. We searched for genes that regulate mitochondrial Ca2+ and H+ concentrations using a genome-wide Drosophila RNA interference (RNAi) screen. The mammalian homolog of one Drosophila gene identified in the screen, Letm1, was found to specifically mediate coupled Ca2+/H+ exchange. RNAi knockdown, overexpression, and liposome reconstitution of the purified Letm1 protein demonstrate that Letm1 is a mitochondrial Ca2+/H+ antiporter.
Yeast genetics and in vitro biochemical analysis have identified numerous genes involved in protein secretion. As compared with yeast, however, the metazoan secretory pathway is more complex and many mechanisms that regulate organization of the Golgi apparatus remain poorly characterized. We performed a genome-wide RNA-mediated interference screen in a Drosophila cell line to identify genes required for constitutive protein secretion. We then classified the genes on the basis of the effect of their depletion on organization of the Golgi membranes. Here we show that depletion of class A genes redistributes Golgi membranes into the endoplasmic reticulum, depletion of class B genes leads to Golgi fragmentation, depletion of class C genes leads to aggregation of Golgi membranes, and depletion of class D genes causes no obvious change. Of the 20 new gene products characterized so far, several localize to the Golgi membranes and the endoplasmic reticulum.
Francisella tularensis is a highly infectious facultative intracellular bacterium that can be transmitted between mammals by arthropod vectors. Similar to many other intracellular bacteria that replicate within the cytosol, such as Listeria, Shigella, Burkholderia, and Rickettsia, the virulence of F. tularensis depends on its ability to modulate biogenesis of its phagosome and to escape into the host cell cytosol where it proliferates. Recent studies have identified the F. tularensis genes required for modulation of phagosome biogenesis and escape into the host cell cytosol within human and arthropod-derived cells. However, the arthropod and mammalian host factors required for intracellular proliferation of F. tularensis are not known. We have utilized a forward genetic approach employing genome-wide RNAi screen in Drosophila melanogaster-derived cells. Screening a library of approximately 21,300 RNAi, we have identified at least 186 host factors required for intracellular bacterial proliferation. We silenced twelve mammalian homologues by RNAi in HEK293T cells and identified three conserved factors, the PI4 kinase PI4KCA, the ubiquitin hydrolase USP22, and the ubiquitin ligase CDC27, which are also required for replication in human cells. The PI4KCA and USP22 mammalian factors are not required for modulation of phagosome biogenesis or phagosomal escape but are required for proliferation within the cytosol. In contrast, the CDC27 ubiquitin ligase is required for evading lysosomal fusion and for phagosomal escape into the cytosol. Although F. tularensis interacts with the autophagy pathway during late stages of proliferation in mouse macrophages, this does not occur in human cells. Our data suggest that F. tularensis utilizes host ubiquitin turnover in distinct mechanisms during the phagosomal and cytosolic phases and phosphoinositide metabolism is essential for cytosolic proliferation of F. tularensis. Our data will facilitate deciphering molecular ecology, patho-adaptation of F. tularensis to the arthropod vector and its role in bacterial ecology and patho-evolution to infect mammals.