6 Papers found (0.009 seconds)

Decker, Johannes H et al. (2009) International Worm Meeting "Identifying host factors required for Sindbis virus replication using C. elegans.."

MacDonald, Margaret, Bradley, Ellen, Decker, Johannes H, Shaham, Shai, Rice, Charles

[

International Worm Meeting,

2009]

Alphaviruses cause equine and human illness characterized by fever, rash, arthritis, encephalitis and even death. Recent outbreaks of an illness resembling dengue fever, caused by the alphavirus Chikungunya, underscore the potential of these viruses to inflict human suffering. Sindbis virus is a well studied Alphavirus. While much is known about viral factors important for Sindbis replication, little is understood about roles played by host factors. To uncover host factors important for Sindbis replication, we generated a transgene containing a heat-inducible promoter (HIP) fused to DNA encoding a modified Sindbis virus, in which all structural proteins were replaced by GFP. GFP expression from this transgene only occurs following viral replication. The HIP::Sindbis(GFP) transgene was introduced and integrated, together with a HIP::mCherry reporter, into wild-type animals. Animals subjected to a 45 min heat shock at 34C express mCherry within 50 min, whereas GFP is seen only after 6 hours, consistent with viral replication. Furthermore, a ribonuclease protection assay of RNA from heat-shocked animals suggests generation of a replication-dependent subgenomic viral fragment containing GFP. Although evident, expression of GFP from the HIP::Sindbis transgene is variable, and of low intensity. We reasoned that since the virus passes through a dsRNA replication intermediate, that low GFP expression may be due to degradation by RNAi. Indeed, animals lacking rde-4, a gene essential for RNAi, express GFP more consistently and at higher levels. GFP was expressed robustly only in muscle cells of the pharynx, vulva, uterus, and body wall. Remarkably, Sindbis replication in vertebrates also exhibits muscle tropism, suggesting that C. elegans may be a suitable model for studying host components promoting replication. To uncover such host factors, we performed EMS mutagenesis on rde-4; HIP-Sindbis(GFP) animals, and screened for reduced GFP expression. We identified 33 mutants, 12 of which retained a functional heat-shock response, and were viable and fertile. We are cloning the genes affected in these mutants. In parallel, we are also crossing our rde-4; HIP-Sindbis(GFP) line to muscle-defective mutants to examine whether genes required for muscle function are required for viral replication. Similar approaches to study replication of other viruses in C. elegans have been previously described, however, it is not yet clear whether host factors described in those studies are required for viral replication in native hosts. Our discovery that Sindbis exhibits similar tropism for replication in C. elegans and vertebrates suggests, perhaps, that similar host factors are indeed involved.

Laha, Michael et al. (2011) International Worm Meeting "Identifying host factors required for Sindbis virus replication using C. elegans."

Decker, Hugo, Bradley, Ellen, Laha, Michael, Rice, Charles, MacDonald, Margaret, Shaham, Shai

[

International Worm Meeting,

2011]

Members of the Alphavirus genus infect equine and human hosts and cause symptoms such as rash, arthritis, fever, encephalitis and in some cases death. Of the Alphaviruses, Sindbis virus (SIN) is the most extensively studied and viral factors required for SIN genome replication have been characterized. While proteomic approaches have uncovered host factors that bind viral proteins, the absence of a genetic model has hindered progress in uncovering the functional roles of such factors. Although a natural C. elegans virus has not been described, a number of viruses have been reported to replicate in C. elegans, suggesting that this animal may provide a suitable setting to study host factors required for SIN replication. To study SIN replication, we generated transgenic C. elegans harboring a SIN replicon in which the structural proteins open reading frame was replaced with GFP, and whose expression is controlled by the heat-shock promoter (HSP). GFP expression from similar SIN replicons in other systems is dependent on viral replication and expression of a subgenomic viral RNA. We found that upon heat shock, transgenic animals expressed low and variable levels of GFP. Introducing the SIN transgene into rde-4 RNAi-defective mutants greatly enhanced expression as assessed by GFP and by Northern analysis, suggesting that the double-stranded RNA SIN replication intermediate is likely inactivated by the RNAi machinery. Although HIP::mCherry co-injected with the HIP::SIN transgene was expressed in all cells upon heat shock, GFP expression was primarily detected in C. elegans muscle cells. This result suggests that SIN preferentially replicates in this cell type. Similar muscle-cell replication tropism has been described for SIN in murine models, and suggests that C. elegans may indeed be an appropriate setting for uncovering host factors required for SIN replication. To uncover such host factors, we performed EMS mutagenesis and isolated 12 mutants with reduced GFP expression. We are using SNP mapping, whole genome sequencing and fosmid rescue experiments to identify the relevant genes. Once identified, genes with obvious mammalian homologs will be studied for roles in established cell culture systems. Our studies may provide insight into the complicated host-Alphavirus interplay that leads to infection and disease.

Gupta, Pratyush et al. (2013) International Worm Meeting "MRG-1 and RFP-1 regulate proliferation in the germline."

Leahul, Lindsay, Gupta, Pratyush, Hansen, David, Jasper, Katie

[

International Worm Meeting,

2013]

We previously demonstrated that the proteasome inhibits the proliferative fate in the C. elegans germ line. Reduced proteasomal function enhances over-proliferation in a sensitized genetic background (Macdonald, Knox et al. 2008). Presumably, the proteasome is involved in degrading proteins that either promote the proliferative fate, or inhibit meiotic entry. We refer to these as proliferation promoting proteins (PPPs). To identify these PPPs, we first sought to identify the substrate recognition subunits (SRS) of E3 ubiquitin ligases that may target the PPPs for degradation by the proteasome. We screened 826 SRSs by RNAi in four sensitized genetic backgrounds and found five that enhance over-proliferation. One of these, RFP-1, specifically enhances glp-1(ar202gf) to form a germline tumor, albeit incomplete. Large-scale 2-hybrid screens identified potential binding partners for RFP-1 (Crowe and Candido 2004; Zhong and Sternberg 2006), which could be PPPs that are targeted for proteasomal degradation by RFP-1. One of these, mrg-1, partially suppresses the over-proliferation phenotype of glp-1(ar202gf); rfp-1(ok572), suggesting that it may function as a PPP. We found that MRG-1 levels do increase in an rfp-1 mutant, as well as when proteasomal function is decreased through genetic mutation or chemical inhibition. Therefore, MRG-1 protein levels are likely regulated through proteasomal-mediated degradation. We are currently determining how mrg-1 may participate in controlling the proliferative fate in the germline.

Burns, Andrew et al. (2021) International Worm Meeting "Selective Control of Parasitic Nematodes with Bioactivated Nematicides"

[

International Worm Meeting,

2021]

Global food security is threatened as the world amasses 10 billion people amid limited arable land. While nematode pests are a major barrier to agricultural intensification, most traditional nematicides are now banned because of poor nematode-selectivity, leaving farmers with inadequate controls. Here, we describe a screen carried out in the model nematode Caenorhabditis elegans that enriches for selective nematicides by identifying molecules that are bioactivated by cytochrome P450s, which are phylogenetically diverse. We identify a family of structures, called nemactivins, that are robustly bioactivated to a toxic metabolite selectively in nematodes. At low parts-per-million concentrations, nemactivins perform comparably well with commercial nematicides at controlling infection by the world's most destructive plant-parasitic nematode Meloidogyne incognita. Hence, nemactivins are first-in-class bioactivated nematicides that provide much needed nematode-selectivity.

GUPTA, P. et al. (2015) International Worm Meeting "MRG-1 and RFP-1 regulate proliferation in the germline."

Hansen, D., Wang, X., GUPTA, P., LEAHUL, L., WANG, C.

[

International Worm Meeting,

2015]

The germ line in C. elegans is organized in an assembly line fashion, with each region containing cells at a specific point in development. As cells move proximally, they progress further through the developmental stages of gametogenesis. Presumably, different factors are expressed in each region to execute each developmental stage. As cells move proximally, some factors must be turned on, while others are turned off. We previously demonstrated that proteasomal degradation of proteins is key for cells to properly exit the proliferative fate and enter into meiotic prophase. Reduced proteasomal function enhances over-proliferation in a sensitized genetic background (Macdonald et al. 2008). We hypothesized that the proteasome is involved in degrading proteins that either promote the proliferative fate, or inhibit meiotic entry. We refer to these as proliferation promoting proteins (PPPs). To understand how proteasomal degradation contributes to maintaining the balance between proliferation and differentiation, we sought to identify these PPPs. To do this, we first focused on identifying the substrate recognition subunits (SRSs) of the E3 ubiquitin ligases to find those that may target the PPPs for degradation. Since the SRSs directly bind their target proteins, we reasoned that if we genetically identify the SRSs involved, we could then biochemically identify the PPPs as interacting proteins. We screened 826 SRSs by RNAi in four sensitized genetic backgrounds and found five proteins that enhance over-proliferation when their activities are reduced. One of these, RFP-1 (putative E3 ligase), enhances glp-1(ar202gf) to form a germline tumor, albeit incomplete. To identify the target proteins of RFP-1, and potential PPPs, we screened the interacting proteins of RFP-1 and looked for suppression of the germline tumor when their activity was reduced. Through this, we identified the chromo-domain containing protein MRG-1. mrg-1(0) partially suppresses over-proliferation in glp-1(ar202gf); rfp-1(ok572) animals, suggesting that it may function as a PPP. We found that MRG-1 levels increase in an rfp-1 mutant, as well as when proteasomal function is decreased through genetic mutation or chemical inhibition, suggesting that its activity is regulated, at least in part, through its levels of accumulation that is likely controlled through proteasomal-mediated degradation (Gupta et al. 2015). Analysis in tissue culture cells further supports that RFP-1 directly targets MRG-1 for degradation by the proteasome. Therefore, we have identified MRG-1 as a key player in regulating the proliferation vs. differentiation decision and determined that its activity is controlled, at least in part, through proteasomal degradation.

Neri C et al. (1998) European Worm Meeting "A C. elegans model of neuron dysfunction in polyglutamine expansion diseases: suggestive evidence for polyglutamine-dependent intracellular aggregation and touch receptor neuron dysfunction"

Chalfie M, Moriniere S, Neri C, Du H-P

[

European Worm Meeting,

1998]

Polyglutamine expansion (PGE) underlies several neurodegenerative diseases including the Huntington!s disease (HD). An inverse correlation between the age-of-onset and the length of the polyglutamine (polyQ) tract is observed in PGE disease families. The mechanisms which cause neurodegeneration in PGE diseases is unknown. Data from human patients, transgenic mice studies, and mammalian cell-based models have suggested that a gain of toxic properties is likely to be induced by PGE in target neurons, resulting in the formation of intracellular aggregates and cell death. Studies of HD in mammalian cell-based models have suggested that both the length of the polyQ tract and the length of the disease protein fragment carrying the expanded polyQ tract influence the localization of intracellular aggregates and the progression of neurodegeneration. The aim of our study is to develop models of PGE-induced neurodegeneration in C. elegans since the genetics, well-characterized development, and the known genomic sequence make this a useful organism in which to study these problems. We used the mec-3 promoter (mec-3p) to express GFP fusions which contain a short N-terminal fragment of huntingtin with a normal (17 units) or expanded (84 units) polyQ tract. The mec-3 gene is expressed in 10 neurons including the six touch receptor neurons AVM, ALML, ALMR, PVM, PLML and PLMR. Transgenic worms were analyzed for intracellular pattern of GFP expression, touch sensitivity, and occurrence of cell death in touch receptor neurons. Mec-3p driven GFP expression typically shows a diffuse expression pattern in the cell body and process. In transgenic animals expressing the long polyQ construct, obvious protein aggregation was observed, particularly as two large and bright perinuclear GFP aggregates were detected. A diffuse GFP expression was never observed in the processes. In transgenic animals expressing the short polyQ construct, a less severe modification of GFP localization was observed, resulting in a pattern of expression closer to that of wild type animals with GFP expression driven by mec-3p. Touch tests were performed over a period of seven days starting from the young adult stage. In worms expressing the long polyQ construct, less than 30% were Mec at the tail at Day 1, and more than 50% were Mec at the tail at Day 7. In worms expressing a short polyQ construct or in animals with GFP expression driven by mec-3p, less than 7% and 30% were Mec at the tail at Day 1 and Day 7, respectively. Cell death was not observed in these experiments. Other studies have suggested that huntingtin fragments with an expanded polyQ tract (150 Glns) induce a neuronal phenotype in transgenic animals (P. Faber, J. Alter, M. MacDonald, and A. Hart, personal communication). Our preliminary data suggest that GFP fusions comprising a short N-terminal huntingtin fragment with 84 consecutive Glns induce perinuclear aggregation and touch insensitivity when expressed in touch receptor neurons. Our data also suggest that the transgenic animals obtained might be useful for studying the biochemical mechanisms involved in polyQ-induced neuron dysfunction.