A Drosophila In Vivo Screen Identifies Store-Operated Calcium Entry as a Key Regulator of Adiposity

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线虫全基因组RNAi筛选确定CAB-1为特异性调节致密核心囊泡分泌的因子

内分泌细胞和神经细胞通过释放激素和神经肽类物质来响应外界刺激,而这些物质的分泌,都是通过致密核心囊泡(dense core vesicle,DCV)来实现的.但是,现阶段关于DCV的生成、转运、释放的机制很大程度上是不清楚的.在本研究中,我们将线虫的排便行为和肠道分泌联系起来,并以此表型进行全基因组RNAi筛选,寻找调节DCV的新基因.我们成功筛选到了一些在肠道调节DCV生成或释放的基因.其中,CAB-1被确认为特异性调节DCV分泌的重要因子.在肠道中,cab-1突变会降低肠道DCV内容物的分泌,而在神经系统中,CAB-1的缺失也会导致DCV的标识物堆积在突触前,而突触囊泡(synaptic vesicle,SV)不受影响.     

An In Vivo C. elegans Model System for Screening EGFR-Inhibiting Anti-Cancer Drugs

The epidermal growth factor receptor (EGFR) is a well-established target for cancer treatment. EGFR tyrosine kinase (TK) inhibitors, such as gefinitib and erlotinib, have been developed as anti-cancer drugs. Although non-small cell lung carcinoma with an activating EGFR mutation, L858R, responds well to gefinitib and erlotinib, tumors with a doubly mutated EGFR, T790M-L858R, acquire resistance to these drugs. The C. elegans EGFR homolog LET-23 and its downstream signaling pathway have been studied extensively to provide insight into regulatory mechanisms conserved from C. elegans to humans. To develop an in vivo screening system for potential cancer drugs targeting specific EGFR mutants, we expressed three LET-23 chimeras in which the TK domain was replaced with either the human wild-type TK domain (LET-23::hEGFR-TK), a TK domain with the L858R mutation (LET-23::hEGFR-TK[L858R]), or a TK domain with the T790M-L858R mutations (LET-23::hEGFR-TK[T790M-L858R]) in C. elegans vulval cells using the let-23 promoter. The wild-type hEGFR-TK chimeric protein rescued the let-23 mutant phenotype, and the activating mutant hEGFR-TK chimeras induced a multivulva (Muv) phenotype in a wild-type C. elegans background. The anti-cancer drugs gefitinib and erlotinib suppressed the Muv phenotype in LET-23::hEGFR-TK[L858R]-expressing transgenic animals, but not in LET-23::hEGFR-TK[T790M-L858R] transgenic animals. As a pilot screen, 8,960 small chemicals were tested for Muv suppression, and AG1478 (an EGFR-TK inhibitor) and U0126 (a MEK inhibitor) were identified as potential inhibitors of EGFR-mediated biological function. In conclusion, transgenic C. elegans expressing chimeric LET-23::hEGFR-TK proteins are a model system that can be used in mutation-specific screens for new anti-cancer drugs.     

A Synthetic Lethal Screen Identifies a Role for Lin-44/Wnt in C. elegans Embryogenesis

BackgroundThe C. elegans proteins PTP-3/LAR-RPTP and SDN-1/Syndecan are conserved cell adhesion molecules. Loss-of-function (LOF) mutations in either ptp-3 or sdn-1 result in low penetrance embryonic developmental defects. Work from other systems has shown that syndecans can function as ligands for LAR receptors in vivo. We used double mutant analysis to test whether ptp-3 and sdn-1 function in a linear genetic pathway during C. elegans embryogenesis.ResultsWe found animals with LOF in both sdn-1 and ptp-3 exhibited a highly penetrant synthetic lethality (SynLet), with only a small percentage of animals surviving to adulthood. Analysis of the survivors demonstrated that these animals had a synergistic increase in the penetrance of embryonic developmental defects. Together, these data strongly suggested PTP-3 and SDN-1 function in parallel during embryogenesis. We subsequently used RNAi to knockdown ~3,600 genes predicted to encode secreted and/or transmembrane molecules to identify genes that interacted with ptp-3 or sdn-1. We found that the Wnt ligand, lin-44, was SynLet with sdn-1, but not ptp-3. We used 4-dimensional time-lapse analysis to characterize the interaction between lin-44 and sdn-1. We found evidence that loss of lin-44 caused defects in the polarization and migration of endodermal precursors during gastrulation, a previously undescribed role for lin-44 that is strongly enhanced by the loss of sdn-1.ConclusionsPTP-3 and SDN-1 function in compensatory pathways during C. elegans embryonic and larval development, as simultaneous loss of both genes has dire consequences for organismal survival. The Wnt ligand lin-44 contributes to the early stages of gastrulation in parallel to sdn-1, but in a genetic pathway with ptp-3. Overall, the SynLet phenotype provides a robust platform to identify ptp-3 and sdn-1 interacting genes, as well as other genes that function in development, yet might be missed in traditional forward genetic screens.     

A Lipid Droplet-Associated GFP Reporter-Based Screen Identifies New Fat Storage Regulators in C.elegans

Fat storage disorders including obesity are pandemic human health problems. As a genetically amenable model organism, Caeno- rhabditis elegans has often been used to explore the molecular mechanisms of fat storage regulation. Dye staining of fixed animals and stimulated Raman scattering （SRS） microscopy methods have been used successfully to study fat storage, but a genetic screening system that takes full advantage of C. elegans transparency to perform live imaging of fluorescent protein reporters has not yet been reported. Here, we investigated the tissue-specific expression of the GFP fusion of Perilipin 1 （PLIN1）, a Drosophila lipid droplet-associated protein, in C. elegans. Our results indicate that PLINI：：GFP labels lipid droplets and can be used as a fat storage indicator in live worms. Through an RNAi screen, we further identified several previously uncharacterized new fat storage regulators.     

Comparative Genomics RNAi Screen Identifies Eftud2 as a Novel Regulator of Innate Immunity

The extent of the innate immune response is regulated by many positively and negatively acting signaling proteins. This allows for proper activation of innate immunity to fight infection while ensuring that the response is limited to prevent unwanted complications. Thus mutations in innate immune regulators can lead to immune dysfunction or to inflammatory diseases such as arthritis or atherosclerosis. To identify novel innate immune regulators that could affect infectious or inflammatory disease, we have taken a comparative genomics RNAi screening approach in which we inhibit orthologous genes in the nematode Caenorhabditis elegans and murine macrophages, expecting that genes with evolutionarily conserved function also will regulate innate immunity in humans. Here we report the results of an RNAi screen of approximately half of the C. elegans genome, which led to the identification of many candidate genes that regulate innate immunity in C. elegans and mouse macrophages. One of these novel conserved regulators of innate immunity is the mRNA splicing regulator Eftud2, which we show controls the alternate splicing of the MyD88 innate immunity signaling adaptor to modulate the extent of the innate immune response.     

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.     

Growth control by EGF repeats of the C. elegans Fibulin-1C isoform

Fibulin is a broadly conserved component of the extracellular matrix (ECM). Previous studies have shown that Caenorhabditis elegans FIBULIN-1 (FBL-1) controls the width of the gonad (Hesselson, D., C. Newman, K.W. Kim, and J. Kimble. 2004. Curr. Biol. 14:2005-2010; Kubota, Y., R. Kuroki, and K. Nishiwaki. 2004. Curr. Biol. 14:2011-2018; Muriel, J.M., C. Dong, H. Hutter, and B.E. Vogel. 2005. Development. 132: 4223-4234). In this study, we report that FBL-1 also controls developmental growth and that one isoform of fibulin-1, called FBL-1C, controls both functions by distinct mechanisms. A large FBL-1C fragment, including both epidermal growth factor (EGF) and fibulin-type C domains, is responsible for constraining gonadal width, but a much smaller fragment containing only two complete EGF repeats (EGF1-2C+) is critical for developmental growth. We suggest that the larger fragment serves a scaffolding function to stabilize the basement membrane and that the smaller fragment provides a regulatory function at the cell surface or within the ECM to control growth.     

The C. elegans ezrin-radixin-moesin protein ERM-1 is necessary for apical junction remodelling and tubulogenesis in the intestine

Members of the ezrin-radixin-moesin (ERM) family of proteins have been found to serve as linkers between membrane proteins and the F-actin cytoskeleton in many organisms. We used RNA interference (RNAi) approach to assay ERM proteins of the Caenorhabditis elegans genome for a possible involvement in apical junction (AJ) assembly or positioning. We identify erm-1 as the only ERM protein required for development and show, by multiple RNA interference, that additional four-point one, ezrin-radixin-moesin (FERM) domain-containing proteins cannot compensate for the depletion of ERM-1. ERM-1 is expressed in most if not all cells of the embryo at low levels but is upregulated in epithelia, like the intestine. ERM-1 protein co-localizes with F-actin and the intermediate filament protein IFB-2 at the apical cell cortex. ERM-1 depletion results in intestine-specific phenotypes like lumenal constrictions or even obstructions. This phenotype arises after epithelial polarization of intestinal cells and can be monitored using markers of the apical junction. We show that the initial steps of epithelial polarization in the intestine are not affected in erm-1(RNAi) embryos but the positioning of apical junction proteins to an apico-lateral position arrests prematurely or fails, resulting in multiple obstructions of the intestinal flow after hatching. Mechanistically, this phenotype might be due to an altered apical cytoskeleton because the apical enrichment of F-actin filaments is lost specifically in the intestine. ERM-1 is the first protein of the apical membrane domain affecting junction remodelling in C. elegans. ERM-1 interacts genetically with the catenin-cadherin system but not with the DLG-1 (Discs large)-dependent establishment of the apical junction.     

Yeast Three-Hybrid Screen Identifies TgBRADIN/GRA24 as a Negative Regulator of Toxoplasma gondii Bradyzoite Differentiation

Differentiation of the protozoan parasite Toxoplasma gondii into its latent bradyzoite stage is a key event in the parasite’s life cycle. Compound 2 is an imidazopyridine that was previously shown to inhibit the parasite lytic cycle, in part through inhibition of parasite cGMP-dependent protein kinase. We show here that Compound 2 can also enhance parasite differentiation, and we use yeast three-hybrid analysis to identify TgBRADIN/GRA24 as a parasite protein that interacts directly or indirectly with the compound. Disruption of the TgBRADIN/GRA24 gene leads to enhanced differentiation of the parasite, and the TgBRADIN/GRA24 knockout parasites show decreased susceptibility to the differentiation-enhancing effects of Compound 2. This study represents the first use of yeast three-hybrid analysis to study small-molecule mechanism of action in any pathogenic microorganism, and it identifies a previously unrecognized inhibitor of differentiation in T. gondii. A better understanding of the proteins and mechanisms regulating T. gondii differentiation will enable new approaches to preventing the establishment of chronic infection in this important human pathogen.     

A Forward Genetic Screen in Zebrafish Identifies the G-Protein-Coupled Receptor CaSR as a Modulator of Sensorimotor Decision Making

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An AGEF-1/Arf GTPase/AP-1 Ensemble Antagonizes LET-23 EGFR Basolateral Localization and Signaling during C. elegans Vulva Induction

LET-23 Epidermal Growth Factor Receptor (EGFR) signaling specifies the vulval cell fates during C. elegans larval development. LET-23 EGFR localization on the basolateral membrane of the vulval precursor cells (VPCs) is required to engage the LIN-3 EGF-like inductive signal. The LIN-2 Cask/LIN-7 Veli/LIN-10 Mint (LIN-2/7/10) complex binds LET-23 EGFR, is required for its basolateral membrane localization, and therefore, vulva induction. Besides the LIN-2/7/10 complex, the trafficking pathways that regulate LET-23 EGFR localization have not been defined. Here we identify vh4, a hypomorphic allele of agef-1, as a strong suppressor of the lin-2 mutant Vulvaless (Vul) phenotype. AGEF-1 is homologous to the mammalian BIG1 and BIG2 Arf GTPase guanine nucleotide exchange factors (GEFs), which regulate secretory traffic between the Trans-Golgi network, endosomes and the plasma membrane via activation of Arf GTPases and recruitment of the AP-1 clathrin adaptor complex. Consistent with a role in trafficking we show that AGEF-1 is required for protein secretion and that AGEF-1 and the AP-1 complex regulate endosome size in coelomocytes. The AP-1 complex has previously been implicated in negative regulation of LET-23 EGFR, however the mechanism was not known. Our genetic data indicate that AGEF-1 is a strong negative regulator of LET-23 EGFR signaling that functions in the VPCs at the level of the receptor. In line with AGEF-1 being an Arf GEF, we identify the ARF-1.2 and ARF-3 GTPases as also negatively regulating signaling. We find that the agef-1(vh4) mutation results in increased LET-23 EGFR on the basolateral membrane in both wild-type and lin-2 mutant animals. Furthermore, unc-101(RNAi), a component of the AP-1 complex, increased LET-23 EGFR on the basolateral membrane in lin-2 and agef-1(vh4); lin-2 mutant animals. Thus, an AGEF-1/Arf GTPase/AP-1 ensemble functions opposite the LIN-2/7/10 complex to antagonize LET-23 EGFR basolateral membrane localization and signaling.     

Sli-1, a Negative Regulator of Let-23-Mediated Signaling in C. Elegans

By screening for suppressors of hypomorphic mutations of let-23, a receptor tyrosine kinase necessary for vulval induction in Caenorhabditis elegans, we recovered >/=12 mutations defining the sli-1 (suppressor of lineage defect) locus. sli-1 mutations suppress four of five phenotypes associated with hypomorphic alleles of let-23 but do not suppress let-23 null alleles. Thus, a sli-1 mutation does not bypass the requirement for functional let-23 but rather allows more potent LET-23-dependent signaling. Mutations at the sli-1 locus are otherwise silent with respect to vulval differentiation and cause only a low-penetrance abnormal head phenotype. Mutations at sli-1 also suppress the vulval defects but not other defects associated with mutations of sem-5, whose product likely interacts with LET-23 protein during vulval induction. Mutations at sli-1 suppress lin-2, lin-7 and lin-10 mutations but only partially suppress lin-3 and let-60 mutations and do not suppress a lin-45 mutation. The sli-1 locus displays dosage sensitivity: severe reduction of function alleles of sli-1 are semidominant suppressors; a duplication of the sli-1 (+) region enhances the vulvaless phenotype of hypomorphic mutations of let-23. We propose that sli-1 is a negative regulator that acts at or near the LET-23-mediated step of the vulval induction pathway. Our analysis suggests that let-23 can activate distinct signaling pathways in different tissues: one pathway is required for vulval induction; another pathway is involved in hermaphrodite fertilty and is not regulated by sli-1.     

A Global In Vivo Drosophila RNAi Screen Identifies a Key Role of Ceramide Phosphoethanolamine for Glial Ensheathment of Axons

Glia are of vital importance for all complex nervous system. One of the many functions of glia is to insulate and provide trophic and metabolic support to axons. Here, using glial-specific RNAi knockdown in Drosophila, we silenced 6930 conserved genes in adult flies to identify essential genes and pathways. Among our screening hits, metabolic processes were highly represented, and genes involved in carbohydrate and lipid metabolic pathways appeared to be essential in glia. One critical pathway identified was de novo ceramide synthesis. Glial knockdown of lace, a subunit of the serine palmitoyltransferase associated with hereditary sensory and autonomic neuropathies in humans, resulted in ensheathment defects of peripheral nerves in Drosophila. A genetic dissection study combined with shotgun high-resolution mass spectrometry of lipids showed that levels of ceramide phosphoethanolamine are crucial for axonal ensheathment by glia. A detailed morphological and functional analysis demonstrated that the depletion of ceramide phosphoethanolamine resulted in axonal defasciculation, slowed spike propagation, and failure of wrapping glia to enwrap peripheral axons. Supplementing sphingosine into the diet rescued the neuropathy in flies. Thus, our RNAi study in Drosophila identifies a key role of ceramide phosphoethanolamine in wrapping of axons by glia.