Yuen-Yi Tseng et al. (2007) International Worm Meeting "Caenorhabditis elegans expresses a functional ArsA ATPase."

Vivian Liao, Chan-Wei Yu, Jui-Tung Liu, Yuen-Yi Tseng

[International Worm Meeting, 2007]

As arsenic is the most prevalent environmental toxin, it is imperative for us to understand the mechanisms of metalloid detoxification. In prokaryotes, arsenic detoxification is accomplished by chromosomal and plasmid-borne operon-encoded efflux systems. The bacterial ArsA ATPase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenite (As(III)) and antimonite (Sb(III)). Herein, we describe the identification of a Caenorhabditis elegans homologue (asna-1) that encodes the ATPase component of the Escherichia coli As(III) and Sb(III) transporter. We have evaluated the responses of wild-type and asna-1 mutant nematodes to various metal ions and found that asna-1 mutant nematodes are more sensitive to As(III) and Sb(III) toxicity than that of wild-type animals. These results provide evidence that ASNA-1 is required for C. elegans'' defense against As(III) and Sb(III) toxicity. A purified maltose-binding protein (MBP)- ASNA-1 fusion protein was biochemically characterized, and its properties were compared with those of ArsAs. ATPase activity of the ASNA-1 protein was dependent on the presence of As(III) or Sb(III). As(III) stimulated ATPase activity by 2 ± 0.2 (SEM) fold, while Sb(III) stimulated it by 4.6 ± 0.15 (SEM) fold. The results indicate that As(III)- and Sb(III)-stimulated ArsA ATPase activities are not restricted to bacteria but extend to animals by demonstrating that the asna-1 gene of the nematode, C. elegans, encodes a functional ArsA ATPase whose activity is stimulated by As(III) and Sb(III) and which is critical for As(III) and Sb(III) tolerance in the intact organism.

M Tijsterman et al. (1999) International C. elegans Meeting "A genetic approach to identify the molecular target of SB-204269 in Caenorhabditis elegans"

M Tijsterman, GP Livi, J Ahringer, RH Plasterk, S Rastan, H Herdon

[International C. elegans Meeting, 1999]

The compound SB-204269 shows potent anticonvulsant activity in a range of animal seizure models without any neurological or cardiovasular side-effects 1 . A stereospecific binding site has been identified in the rat brain 2 but the molecular nature of this unique binding site remains to be elucidated. We have started to use the strength of C. elegans genetics in an attempt to identify the molecular target of SB-204269. Exposure of mothers to SB-204269 (micromolar range) results in embryonic lethality in this organism whereas its enantiomer SB-204268 does not. A C. elegans strain with a general increased sensitivity to drugs ( pgp-1; pgp-3; mrp-1 ) are also more sensitive to SB-204269 than Bristol N2 and the obtained level of killing allows a large-scale selection for mutants that have gained resistance specifically to the compound. Besides the use of EMS as a source to introduce mutations into the C. elegans genome we crossed a mutator allele ( mut-7 ) into this genetic background to allow a more easy detection of the causal mutation via a transposon display experiment. Both EMS- and "mutator"-screens are currently ongoing. 1 Upton et al. (1997) Br. J. Pharmacol., 121, 1679-1686 2 Herdon et al. (1997) Br. J. Pharmacol. 121, 1687-1691

Siavash Karimzadegan et al. (2007) International Worm Meeting "A mutagenesis screen for genes involved in deactivating the unc-4 transcription factor."

Victor Chiang, Martin Chalfie, Siavash Karimzadegan

[International Worm Meeting, 2007]

Transcriptional activators are often modulated and eventually turned off after their initial activation. While many studies have investigated transcription factor activation pathways, relatively less attention has been paid to the deactivation of these molecules. [Kodadek et al. (2006) Cell 127: 261-264]. Using a short-lived GFP driven by the unc-4 promoter, we have examined the inactivation of this transcription factor. UNC-4 is a homeodomain protein [Miller et al. (1992) Nature 355: 841-845] that is expressed in A-type motorneurons and appears to specify their differentiation including their synaptic partners. Northern blots indicate that peak unc-4 mRNA transcript levels occur at late L1/early L2 [Miller et al. (1995) Development 121: 2877-2886], concurrent with the developmental stage when A-type motorneurons receive synaptic input from interneurons [Miller et al. (1992) Nature 355: 841-845]. Thereafter, transcript levels fall precipitously, and adults no longer express unc-4 mRNA. Similarly, strains carrying unstable GFP driven by the unc-4 promoter produce GFP label in A-type motorneurons only at this L1/L2 stage. [Zhang et al. (2004) Cell 119(1): 137-44; Poyurovsky et al. (2004) Mol Cell 12(4): 875-87]. We have used this strain to screen for mutants in order to identify candidates where the unc-4 promoter is constitutively active beyond the L1/L2 stage. A screen of 22,000 haploid genomes yielded 12 candidates where GFP fluorescence is observed in all A-type motorneurons during adult stage. A further 19 candidates showed GFP fluorescence in a subset of the A-type motorneuron during adult stage. Complementation and mapping are in progress to identify these candidates.

Corey A Morris et al. (2003) International Worm Meeting "Site-directed mutagenesis of the transcription factor LIN-31 reveals functional domains required in the regulation of vulval cell fates."

David B Doroquez, E Lorena Mora-Blanco, Leilani M Miller, Yvonne Yang, Corey A Morris

[International Worm Meeting, 2003]

In order to better understand cell fate determination in Caenorhabditis elegans, we are conducting a functional analysis of LIN-31, a winged-helix transcription factor (WH TF) that acts as a tissue-specific effector of the conserved Ras/MAP kinase signaling pathway to promote or suppress vulval cell fates in the development of the hermaphrodite vulva (Miller et al., Genes and Dev., 7:933, 1993). In addition to a DNA-binding domain (DBD), the LIN-31 protein contains several regions of interest: a small acidic-rich region, four MAP kinase consensus phosphorylation sites, and a small region at the C-terminus that displays homology with a subset of WH proteins. These regions could play a number of roles, from transcriptional activation to an interaction domain for LIN-1, which is known to heterodimerize with LIN-31 (Tan et al., Cell, 93:569,1998). Using site-directed mutagenesis techniques, specific mutations were introduced into the gene at these regions of interest. Stable transgenic lines were created through germline microinjection of mutant plasmids into animals with no functional LIN-31. Through phenotypic analysis of multiple transgenic lines, we are beginning to better understand the functional significance and contribution of each of these different sites to LIN-31 function. Our results thus far support the current model (Miller et al., 1993; Tan et al., 1998; Miller et al., Genetics, 156:1595, 2000), that LIN-31 has two functions: 1) to activate vulval cell fates in P5.p, P6.p and P7.p; and 2) to repress vulval cell fates in P3.p, P4.p, and P8.p.In addition, we are initiating an in vitro functional analysis of LIN-31 protein. We used a bacterial expression system to produce GST::LIN-31 fusion protein. Using electrophoretic mobility shift assays, we have determined that wild-type LIN-31 protein is able to specifically bind the promoter of another WH TF target. LIN-31's ability to interact with this promoter was disrupted when 1) LIN-31 carried a previously characterized point mutation in the DBD believed to disrupt its interaction with the target DNA (Miller et al., 2000) and 2) when the promoter sequence contained base substitutions. We are now in the process of creating, expressing, and purifying mutant GST::LIN-31 fusion proteins in order to investigate LIN-31 sequences required for heterodimerization with LIN-1.

Corey A Morris et al. (2002) West Coast Worm Meeting "SITE-DIRECTED MUTANTS OF THE C. elegans TRANSCRIPTION FACTOR LIN-31 REVEAL DISTINCT FUNCTIONS."

Corey A Morris, E Lorena Mora-Blanco, Yvonne Yang, David B Doroquez, Leilani M Miller

[West Coast Worm Meeting, 2002]

In order to better understand cell fate determination in Caenorhabditis elegans, we are conducting a functional analysis of LIN-31, a winged-helix transcription factor (WH TF) that acts as a tissue-specific effector of the conserved Ras/MAP kinase signaling pathway to promote or suppress vulval cell fates in the development of the hermaphrodite vulva (Miller et al., Genes and Dev., 7:933, 1993). In addition to a DNA-binding domain (DBD), the LIN-31 protein contains several regions of interest: a small acidic-rich region, four MAP kinase consensus phosphorylation sites, and a small region at the C-terminus that displays homology with a subset of WH proteins. These regions could play a number of roles, from transcriptional activation to an interaction domain for LIN-1, which is known to heterodimerize with LIN-31 (Tan et al., Cell, 93:569,1998). Using site-directed mutagenesis techniques, specific mutations were introduced into the gene at these regions of interest. Stable transgenic lines were created through germline microinjection of mutant plasmids into animals with no functional LIN-31 protein. Through phenotypic analysis of multiple transgenic lines, we are beginning to better understand the functional significance and contribution of each of these different sites to LIN-31 function. Our results thus far support the current model (Miller et al., 1993; Tan et al., 1998; Miller et al., Genetics, 156:1595, 2000), that LIN-31 has two functions: 1) to activate vulval cell fates in P5.p, P6.p and P7.p; and 2) to repress vulval cell fates in P3.p, P4.p, and P8.p. In addition, we are initiating a functional analysis of LIN-31 protein using two assays: ability to bind a putative DNA target sequence and ability to heterodimerize with LIN-1. We used a bacterial expression system to produce GST::LIN-31 fusion protein. Using gel-shift assays, we confirmed function of wild-type protein by demonstrating its ability to bind the transthyretin (TTR) promoter, a consensus sequence recognized by HNF-3, another WH TF sharing DBD sequence homology (Costa et al., Mol. Cell. Biol., 9:1415, 1989). We are now in the process of creating, expressing, and purifying GST::LIN-31 fusion proteins carrying specific mutations, including two point mutations in the DBD believed to disrupt interaction of the LIN-31 with its target DNA (Miller et al., 2000). These mutant proteins will allow us to test in vitro their ability to bind the TTR promoter and to heterodimerize with LIN-1.

Hayes K et al. (1998) West Coast Worm Meeting "CHARACTERIZATION OF CAENORHABDITIS ELEGANS VULVAL CELL FATE MUTANTS THAT MAY ACT DOWNSTREAM OF LIN-31"

Miller LM, Mahoney S, Hayes K

[West Coast Worm Meeting, 1998]

We are interested in studying how cells choose their fates. An extremely good system for studying cell fate specification is the development of the hermaphrodite vulva in the soil nematode, Caenorhabditis elegans. In this developmental pathway, vulval precursor cells are induced to adopt vulval cell fates via an EGF receptor/ras/MAP kinase phosphorylation cascade. This study will focus on the identification and characterization of new genes in the cell-signaling pathway responsible for vulval development in C. elegans. While the early stage of this pathway is known to comprise many proteins homologous to those involved in mammalian and Drosophila receptor tyrosine kinase signal transduction, later stages are not well-understood. One of the latest-acting genes in this pathway is the transcription factor, LIN-31 fates (Miller et al., Genes and Dev., 7:933, 1993). To identify genes downstream of the lin-31 gene, vulvaless (Vul) mutants were isolated in a lin-31 mutant background (Miller et al, unpublished observations). lm55 and lm104, recessive mutations identified in this screen, both enhance the Vul phenotype of a lin-31 null mutation from 15% Vul to 82% and 85%, respectively.. lm55 has been mapped to a small region of chromosome IV and clearly shows defects in choice of vulval cell fate. lm104 has been mapped to a small region on the right arm of chromosome II. Both mutations are Vul in the absence of a lin-31 mutation, indicating that they play a unique and critical role in vulval development.

Douglas, C.D. et al. (2011) International Worm Meeting "The effects of nickel toxicity upon survival, growth, and reproduction in nematodes."

Ingersoll, C.G., Besser, J.M., Douglas, C.D., Rudel, D.

[International Worm Meeting, 2011]

Nickel is one of the most prevalent heavy metal contaminants in the dust floating in the air we breath, in soils, and in many waterways. Contamination continues to increase largely due to human activities like mining, manufacturing, and waste management. Ni2+ is a carcinogen to plants and animals, and a major concern for the health and safety both of humans and food crops. In addition to mutagenesis and cancer, nickel exposure leads to a variety of other deleterious health conditions. In collaboration with the United States Geological Survey, an assay to evaluate the effects of substrate bound nickel and soluble nickel on survival, growth, and fecundity using C. elegans and Pristionchus pacificus has been developed. Synchronized L1 (C. elegans) or J2 (P. pacificus) larvae were added to sediment and water samples in twelve-well tissue culture plates with a defined amount of freshly killed bacteria as a food source. Animals were grown for 96 hours at 20degC and harvested. Harvested animals were assayed for survival, growth, and fertility. Growth in eight sediment-types collected at discrete uncontaminated sites along the Missouri/Mississippi river basin suggests that both nematodes grow well in organic based sediments and less well in more sandy/inorganic based sediments. Growth in sediments spiked with increasing amounts of environmentally relevant levels of sediment-bound nickel showed that C. elegans was highly sensitive to nickel in the environment of the sediment. Treatment was lethal prior to adulthood. Growth in nickel spiked liquid media with freshly killed food and cholesterol, i.e. hard H20 pH 7.5, distilled (DI) H20, SB Media (hard H20), and SB Media (DI H20), showed that C. elegans has a strong tolerance for environmentally relevant levels of soluble nickel. Adult survivors of nickel treatments showed no significant effects on adult length/width measurements or the presence of fertilized eggs in the uterus. As nickel has other known cell stress effects, we will also assay total germline apoptosis as an measure of DNA damage.

Schultz, Jessica L. et al. (2013) International Worm Meeting "A Complex Issue: Understanding vMSP Receptor Heteromeric Complexing Behavior."

Miller, Michael, Lee, Se-Jin, Schultz, Jessica L., Han, Sung Min

[International Worm Meeting, 2013]

Amyotrophic Lateral Sclerosis (ALS) is a lethal neurodegenerative disease with an unknown pathogenesis and limited therapeutic treatments. A major limitation hindering the development of therapeutic treatments is a lack of understanding of the disease's molecular pathways. In humans, a P56S point mutation in the VAPB/ALS8 MSP domain is associated with ALS and late-onset spinal muscular atrophy (SMA) (Funke et al., 2010; Millecamps et al., 2010; Nishimura et al., 2004). The N-terminal MSP domain is cleaved from the C-terminus of the VAPB protein, and is secreted in a cell-type specific manner (Tsuda et al., 2008). However, the P56S mutation inhibits secretion of the MSP domain. Genetic and biochemical evidence support the hypothesis that the MSP domain interacts with the VAB-1 Eph receptor, ROBO/SAX-3 receptor, and CLR-1 Lar-like protein tyrosine phosphatase receptor, which are collectively called growth cone guidance receptors (Miller et al, 2001; Miller et al., 2003; Tsuda et al., 2008; Han et al., 2012). In C. elegans, secreted vMSP acts on CLR-1 and ROBO/SAX-3 receptors expressed in striated muscle, promoting Arp2/3-dependent actin remodeling. This remodeling is critical for proper placement of mitochondria to actin-rich myofilament I-bands (Han et al., 2012). We hypothesize that the vMSP receptors form heteromeric complexes to promote signaling critical for actin remodeling and correct mitochondria placement. To begin testing this hypothesis, I am expressing combinations of VAB-1, SAX-3, and CLR-1 in cultured cells and investigating putative complex formation via co-immunoprecipitation. My preliminary data suggest that SAX-3 complexes with both VAB-1 and CLR-1. Data will also be presented on the role of C. elegans VAPB/VPR-1 in regulating mitochondria in motor neurons. The results could provide insight into growth cone guidance receptor interactions and pathways involved in ALS.

Eva L Mora-Blanco et al. (2001) International C. elegans Meeting "SITE-DIRECTED MUTAGENESIS OF LIN-31, A WINGED-HELIX TRANSCRIPTION FACTOR INVOLVED IN CAENORHABDITIS ELEGANS VULVAL DEVELOPMENT"

Leilani M Miller, Eva L Mora-Blanco, David B Doroquez, Yvonne Yang, Jill Haggerty

[International C. elegans Meeting, 2001]

In order to understand cell fate specification during vulval development, we are conducting a structure/function analysis of LIN-31, a member of the winged-helix family of transcription factors, which is required for the proper specification of vulval cell fates in C. elegans (Miller et al. , Genes and Dev., 7:933, 1993). The LIN-31 protein contains a DNA-binding domain, an acidic region, four MAP kinase consensus phosphorylation sites, and a small region of homology conserved among other winged-helix proteins. We are using site-directed mutagenesis to create plasmids carrying specific mutations in the MAP kinase consensus phosphorylation sites, acidic domain, or the homology region of the LIN-31 protein. These plasmids are then injected into an animal with no functional LIN-31 protein to test for their ability to provide LIN-31 function. LIN-31 is phosphorylated by the MAP kinase MPK-1 in response to an inductive signaling event (Tan et al. , Cell 93: 569, 1998). Tan et al . have already shown that removing all four MAP kinase consensus phosphorylation sites inactivates one of LIN-31's functions, but the individual contribution of each site is not known. Four individual clones are being created, with each one containing a different disrupted MAP kinase consensus phosphorylation site. In addition, it is appealing to imagine that the small acidic domain (six consecutive aspartic acid residues) adjacent to the DNA-binding domain is functioning as a transcriptional activator, as is the case with acidic regions in some other transcription factors. There is no proof, however, that this rather small acidic domain in LIN-31 is even required for function. Five mutant clones, in which different portions of the acidic domain have been removed or replaced will address this question. Finally, the carboxy-terminus of LIN-31 contains a small region of homology that shows similarity to a subset of winged-helix proteins. The function of this homology region is also unknown and is being explored in this project. This structure/function study is especially appealing since the current model (Miller et al. , 1993; Tan et al. , 1998; and Miller et al. , Genetics 156: 1595, 2000) proposes that LIN-31 has two functions: 1) to activate vulval cell fates in P5.p, P6.p, and P7.p and 2) to repress vulval cell fates in P3.p, P4.p, and P8.p. Microinjection of these clones into lin-31(null) animals will allow us to test if each specific mutation disrupts one, both, or none of LIN-31's functions. Thus, this approach will allow us to identify the roles of specific domains or sites in the LIN-31 protein.

Miller, Rachel K. et al. (2009) International Worm Meeting "CSN-5, a Component of the COP9 Signalosome Complex, Regulates the Levels of UNC-96 and UNC-98, two Components of M-lines in C. elegans muscle."

Mercer, Kristina B., Benian, Guy M., Miller, Rachel K., Wortham, Tesheka S., Stark, Thomas J., Qadota, Hiroshi

[International Worm Meeting, 2009]

UNC-98 and UNC-96 are two small proteins that localize to the M-lines in body wall muscle. Mutants of each gene have a similar and characteristic phenotype: by polarized light microscopy, each shows disorganization of myofibrils and birefringent needles at the ends of the muscle cells. There is genetic and biochemical evidence that UNC-98 and UNC-96 interact with each other (Mercer et al. 2006), and with paramyosin (Mercer et al. 2006; Miller et al. 2008). unc-96 mutants contain discrete accumulations of UNC-98 protein, and unc-98 mutants contain accumulations of UNC-96 protein. Moreover, in both unc-98 and unc-96 mutants, paramyosin is localized both normally to A-bands and abnormally in accumulations. By western blot, in the absence of paramyosin, UNC-98 is diminished, whereas in paramyosin missense mutants, UNC-98 is increased. To explain this and other data, we have proposed a model in which UNC-98 and UNC-96 act as chaperones to promote the incorporation of paramyosin into thick filaments (Miller et al. 2008). We now report that, unexpectedly, both UNC-98 and UNC-96 interact with CSN-5, a component of the conserved COP9 signalosome which has been implicated in a wide variety of functions usually linked to ubiquitin-mediated proteolysis. The interactions were initially found by screening of a yeast 2-hybrid library, and then confirmed by biochemical methods. Anti-CSN-5 antibody co-localized with paramyosin at A-bands in wild type, and co-localized with accumulations of paramyosin in unc-98, unc-96, and unc-15 mutants. Double knock down of csn-5 and the homologous csn-6 could slightly suppress the unc-96 mutant phenotype. In the double knock down of csn-5 and csn-6, the levels of UNC-98 protein were greatly increased and the levels of UNC-96 protein were slightly reduced, suggesting that CSN-5 promotes the degradation of UNC-98 and that CSN-5 stabilizes UNC-96. In unc-15 and unc-96 mutants, CSN-5 protein was reduced, implying the existence of feed back regulation from myofibril proteins to CSN-5 protein levels. Our results are the first to implicate CSN-5 or the COP9 signalosome in myofibrillar organization or function.