Bush JA et al. (1996) West Coast Worm Meeting "THE ROLE OF UNC-52 DOMAIN IV IMMUNOGLOBULIN REPEATS IN MYOFILAMENT ASSEMBLY"

Moerman DG, Rahmani P, Bush JA, Mullen GP

[West Coast Worm Meeting, 1996]

The unc-52(II) gene encodes the nematode homologue of perlecan, a mammalian basement membrane (BM) proteoglycan. UNC-52 is essential for proper myofilament assembly and muscle attachment in C. elegans. The unc-52 gene has 26 exons which, through alternative splicing, generate several UNC-52 isoforms. These isoforms show tissue and temporal specificity (see abstract by Mullen et al). The domain structure of unc-52 lends itself to regulation via alternative splicing. In particular, domain IV of the gene encodes 14 Ig repeats and most of these are contained within individual exons. Alternative splicing of exons 15 - 18 can alter Ig number within this domain. Long isoforms of UNC-52 containing portions of domain IV are localized to BMs underlying body wall muscles in the developing embryo. Shorter isoforms lacking domain IV are found associated with the BMs adjacent to the developing pharynx and anal sphincter/depressor muscles. Null mutations in unc-52 lead to a Pat terminal phenotype (e.g. st549) while mutations in exons 16 -18 lead to an Unc terminal phenotype (e.g. e444). From the sequence analysis of two new unc-52 mutations, st560 and ra112, we conclude that it is domain IV-containing isoforms of unc-52 that are critical for myofilament assembly during early development. These two mutations lead to a Pat terminal phenotype and each affects only domain IV of unc-52; st560 is a stop codon in exon 13; ra112 is an out-of-frame deletion removing several Ig-encoding exons within domain IV. Using a Tc1 excision and double-strand break repair scheme to vary the number of Ig repeats within an isoform, we are attempting to discern the functional significance of domain IV in establishing proper muscle assembly. We are interested in determining the minimal number of Ig repeats necessary within domain IV to allow proper myofilament assembly and to determine the role of combinatorial Ig repeats in assembly. So far we have characterized eight in-frame Tc1 excision deletion alleles which eliminate anywhere from 1 to 4 Ig repeats. Animals homozygous for these deletions appear wild-type in movement and muscle structure.

Mullen GP et al. (1996) West Coast Worm Meeting "UNC-52 ISOFORM DIVERSITY: AN IMMUNOLOGICAL AND GENETIC ANALYSIS"

Bush JA, Mullen GP, Rogalski TM, Moerman DG

[West Coast Worm Meeting, 1996]

Alternative splicing of unc-52 pre-mRNA gives rise to several protein isoforms. These isoforms can be divided into two groups: (i) short forms which consist of domains I-III, and (ii) long forms which consist of all four domains but differ in the number of immunoglobulin-like (Ig) repeats in the fourth domain. The number of Ig-repeats in this group of isoforms is regulated by the mec-8 gene which affects the accumulation of certain alternatively-spliced unc-52 transcripts (Lundquist et al., Development 122 (5), 1996) . Using domain-specific antibodies, we have characterized the localization of UNC-52 isoforms in wild-type and mutant animals. Our results indicate that there are developmental and tissue-specific differences in isoform localization. For example, in embryos short forms are restricted to the pharynx and anal muscles, while the long forms are restricted to the body wall muscles. Our studies on mutants indicate that long isoforms are essential for myofilament assembly in body wall muscles and implicate domain IV in the initial assembly events. To address the significance of structural diversity within domain IV, we have used two strategies to reduce the complexity of Ig motif isoforms. We have identified eight transposon-induced in-frame deletions which remove anywhere from one to four alternatively-spliced exons contributing to domain IV (see abstract by Bush et al.). Animals homozygous for these deletion alleles are viable and have no discernible mutant phenotype suggesting that some UNC-52 isoforms have redundant functions. We have also constructed several mec-8;unc-52 double mutants and characterized these by antibody staining. These animals are missing a subset of long isoforms as a result of the additive effects of mec-8 and unc-52(viable) mutations. Our results indicate that at least one subset of UNC-52 isoforms is restricted to muscle cells in the head region of the embryo.

Mullen GP et al. (1997) International C. elegans Meeting "GENETIC INTERACTIONS BETWEEN unc-52, mec-8, AND lin-14 AND REGULATION OF THE ALTERNATIVE-SPLICING OF unc-52 PRE-mRNA."

Bush JA, Moerman DG, Mullen GP

[International C. elegans Meeting, 1997]

Alternative splicing of unc-52 pre-mRNA gives rise to distinct protein isoforms that exhibit spatial and temporal differences in expression and localization. Recently, mec-8 has been shown to encode a putative RNA-binding protein that regulates some of the alternative splicing of unc-52 pre-mRNA (Lundquist et al. Development 122, 1601-1610). Further studies show that mec-8 has differential effects on the accumulation and distribution of specific UNC-52 isoforms. The lin-14 gene is one of several heterochronic genes that have been identified in C. elegans (Ambros, V. Cell 57, 49-57). We have found that lin-14(loss-of-function) mutations enhance the unc-52(viable) phenotype; lin-14;unc-52 double mutants become paralyzed one larval stage earlier than unc-52(viable) single mutants. The genetic interaction between lin-14 and unc-52 suggests that lin-14 may regulate the developmental timing of certain unc-52 splicing events, perhaps through regulation of mec-8. We are currently examining the developmental profile of certain unc-52 alternative splice events in wild-type and mutant backgrounds to test this prediction. To ask whether alternative splicing is actually required at unc-52, we isolated a number of spontaneous revertants of a Tc1 insertion in exon 18 of unc-52. These deletion revertants are all in-frame events and delete part or all of the alternatively-spliced region. The largest of these, ra515, spans the region from exon 14 to 19 and has a wild-type phenotype even in combination with mec-8(u74), demonstrating that the elimination of alternative-splicing in this region bypasses the need for mec-8, at least with respect to unc-52. This leads us to ask why unc-52 is alternatively spliced in the first place!!!

Mullen GP et al. (1995) International C. elegans Meeting "SPATIAL AND TEMPORAL EXPRESSION OF UNC-52 PROTEIN ISOFORMS AND THEIR ROLE IN MUSCLE ATTACHMENT."

Rogalski TM, Moerman DG, Mullen GP, Bush JA

[International C. elegans Meeting, 1995]

The unc-52 gene produces a number of distinct polypeptides that are found in the basement membranes underlying the body wall muscles, pharynx, gonad and anal sphincter/depressor muscles. To examine the expression of these various gene products, we have generated antibodies to specific regions of domains III (laminin-like) and IV (NCAM-like) of the unc-52 sequence. These include antibodies reactive to a constitutively expressed region, alternative carboxyl termini, and several alternatively spliced exons. Staining with these antibodies has revealed temporal and tissue-specific diferences in isoform localization. For example, isoforms with domain IV are restricted to the basement membranes underlying the body wall muscles during early development. By staining embryos homozygous for Pat alleles of unc-52, we have identified an UNC-52 domain essential for myofilament assembly and attachment during embryogenesis. Whereas some Pat mutants lack all UNC-52 staining, a subset lack only domain IV-containing isoforms. The shorter isoforms are present and properly localized in these embryos. Based on these observations, we propose that perlecan/UNC-52 probably anchors the integrin complex of muscle dense bodies and M-lines through its NCAM domain (domain IV).

Aamna Kaul et al. (2003) International Worm Meeting "Hyperactivation of glutamate-gated chloride channels with ivermectin results in necrosis."

Joseph Dent, Aamna Kaul

[International Worm Meeting, 2003]

Ivermectin is a widely used antiparasitic drug. It kills worms by activating glutamate-gated chloride channels (GluCls), which belong to the family of ligand-gated anion channels that includes the GABA and glutamate receptors (Cully et al., 1994; Dent et al., 2000). The chloride permeability that ivermectin induces in excitable cells tends to prevent excitation. For example, ivermectin targets a GluCl expressed in the pharyngeal muscle to inhibit muscle contraction and prevent eating (Dent et al., 1997). The worms linger for several days in the presence of ivermectin before they starve to death. However, we have found that the lethal effects of ivermectin on C. elegans become irreversible after only a few hours of exposure. When L1 worms were exposed to 20ng/ml for 5 hours and then washed, they gradually developed large vacuoles in their pharyngeal muscle over the next several days. A mutant strain that lacks ivermectin receptors shows little or no necrosis when treated. Ivermectin is hydrophobic and it irreversibly opens GluCls expressed in Xenopus oocytes. So it is possible that ivermectin persists in membranes and continues to activate GluCls. Furthermore, it has been shown that hyperactive cation channels can induce excitotoxic necrosis (Driscoll and Chalfie, 1991). Why, though, would an inhibitory channel have a similar effect when hyperactivated? We are trying to address this question by looking at whether mutations known to inhibit excitotoxicity also inhibit the necrotic effects of ivermectin. Cully DF, Vassilatis DK, Liu KK, Paress PS, Van der Ploeg LHT, Schaeffer JM, Arena JP. Nature 371: 707-711 1994 Dent JA, Smith MM, Vassilatis DK, Avery L. PNAS USA 97: 2674-2679 2000 Dent JA, Davis MW, Avery L. EMBO Journal 16: 5867-5879 1997 Driscoll, M and Chalfie, M. Nature 349: 588-593 1991

Sedore, C. et al. (2019) International Worm Meeting "NDGA and Green Tea Extract reproducibly and robustly increase longevity, as evaluated using an updated Caenorhabditis Intervention Testing Program (CITP) workflow."

Huynh, P., Guo, M., Onken, B., Xue, J., Garrett, T., Harinath, G., Bhaumik, D., Hall, D., Coleman-Hulbert, A., Guo, S., Sedore, C., Lithgow, G., Driscoll, M., Lucanic, M., Chen, E., Johnson, E., Phillips, P., Chen, M., Plummer, T., Foulger, A.

[International Worm Meeting, 2019]

Limiting the debilitating consequences of aging is a major medical challenge of our time, and evaluating pharmacological interventions that promote healthy aging in a robust and reproducible manner across diverse genetic backgrounds remains an important goal in the field. Here, we present the updated experimental workflow for the Caenorhabditis Intervention Testing Program (CITP) for evaluating lifespan and healthspan promoting compounds. We have used this workflow to examine variation in longevity across nine Caenorhabditis strains spanning three species, using multiple replicates collected across three independent laboratories for a set of seven new putative pro-longevity compounds (to add to the ten CITP-tested compounds previously reported). Of the seven tested here, two were dropped from the initial phase of our pipeline due to negative results, while five of the most promising compounds were further evaluated in advanced phases of the workflow. Of these, NDGA, an antioxidant compound derived from the Creosote bush, and Green Tea Extract, a compound extract rich in polyphenols, significantly extended lifespan at multiple concentrations among multiple Caenorhabditis strains. Our updated experimental workflow, combined with the capacity of our multi-institutional consortium, provides an invaluable method for testing lifespan-promoting compounds in an efficient, reproducible, and highly robust manner.

Jiang H et al. (2000) Midwest Worm Meeting "AHA! bHLH-PAS proteins may mediate response to hypoxia in C. elegans"

Jiang H, Guo R, Powell-Coffman JA

[Midwest Worm Meeting, 2000]

O2 homeostasis is essential for the survival of all organisms. From bacteria to mammals, cellular O2 levels are carefully regulated. Low O2 (hypoxia) will endanger ATP generation by shutting down the oxidative phosphorylation. In mammals hypoxia-inducible factor (HIF-1) plays a central role in regulation of responses to low O2 levels. It consists of two bHLH-PAS transcriptional subunits, HIF-1a and HIF-1b. HIF-1b is better known as aryl hydrocarbon receptor nuclear translocator (ARNT). Under hypoxic conditions, HIF-1a protein is stabilized and dimerizes with ARNT to form an active transcription heterodimer (HIF-1), which in turn activates the transcription of downstream targets. The target gene products either promote O2 delivery or switch metabolism to low O2 consumption. Multiple mechanisms of O2 sensing have been proposed, but none have been vigorously tested in vivo (1). Here, we report that bHLH-PAS genes may mediate response to hypoxia in C. elegans. The C. elegans genome encodes 5 putative transcription factors that contain basic-helix-loop-helix and PAS domain motifs. Two of these genes, ahr-1 and aha-1, encode the orthologs of the aryl hydrocarbon receptor and ARNT, respectively (2). Genetic analysis of these two genes has demonstrated that aha-1 is essential for viability, while ahr-1 is not (3,4). This indicates that AHA-1 may dimerize with other bHLH-PAS proteins to regulate developmentally important processes. Four lines of evidence suggest that AHA-1 and F38A6.3a may form hypoxia-responsive complex in C. elegans. First, AHA-1 and the F38A6.3a gene product can be co-immunoprecipitated when expressed in vitro. Second, F38A6.3a mRNA levels increase in hypoxic conditions. Third, we have identified potential target genes that are induced by hypoxia in N2, but not in animals homozygous for a deletion in F38A6.3a. Fourth, AHA-1 is expressed in many tissues (3, 4), and F38A6.3a:GFP is ubiquitously expressed. Further characterization of the mutant phenotype will be presented at the meeting. References: 1. Semenza GL. Perspectives on Oxygen Sensing. Cell (1999) 98(3): 281-4. 2. Powell-Coffman JA, Bradfield CA, Wood WB. Caenorhabditis elegans orthologs of the aryl hydrocarbon receptor and its heterodimerization partner with the aryl hydrocarbon receptor nuclear translocator. PNAS (1998), 95: 2844-2849. 3. Jiang H, Guo R, Powell-Coffman JA. An essential role for aha-1, a PAS-domain-containing regulatory gene in C. elegans. 1999 International Worm Meeting, 438. 4. See other Powell-Coffman lab abstracts for this meeting.

Wang X et al. (1998) East Coast Worm Meeting "A MOLECULE CONTAINING A HEDGEHOG-LIKE AUTOPROCESSING DOMAIN IS REQUIRED FOR MOLTING IN C. ELEGANS."

Beachy P, Seydoux GC, Wang X

[East Coast Worm Meeting, 1998]

The Hedgehog family of signaling molecules controls many aspects of animal development, from embryonic patterning to limb formation. Recently, these proteins have been shown to rely on a novel mechanism to regulate their spatial distribution (1,2). Hedgehog proteins undergo an autoprocessing reaction to yield two distinct products, an amino-terminal fragment (Hh-N), and a carboxy-terminal fragment (Hh-C). Hh-N functions in signaling, whereas Hh-C mediates the processing reaction. This reaction proceeds via an internal thioester intermediate and results in the covalent linkage of cholesterol to Hh-N. This modification in turn causes Hh-N to remain tightly associated with the cell surface, thus effectively limiting its free diffusion and range of action. We are interested in determining whether similar mechanisms may be operating in the biogenesis of other secreted molecules. Towards this goal, we have started to characterize a set of eight C. elegans proteins with sequence similarity to the Hh-C domain of Hedgehog (2,3). In these proteins, as in the Hedgehog family, the Hh-C-like domain is located in the carboxy-teminal end, and is preceded by an amino-terminal domain bearing a signal sequence (but with otherwise no homology to Hh-N). This structure is consistent with the possibility that these proteins are secreted and undergo processing in a manner similar to that described for Hedgehog. To test this possibility we have fused the Hh-C-like domain of one of these proteins to a His tag, and purified the fusion protein from E. coli. We find that such a fusion can undergo the processing reaction in vitro, suggesting that the Hh-C-like domain is functionally active in the nematode proteins. We have begun to characterize the functions of Hh-C-related proteins using RNA-mediated interference. Our initial results suggest that one of these proteins, T05C12, is essential for molting. Injection of T05C12 double-stranded RNA results in 90% larval lethality. The larvae apparently die from a failure to shed old cuticles during molts. In many animals, a cuticular plug can be seen obstructing the mouth opening where it presumably interferes with feeding. The amino-terminal domain of T05C12 contains several sequence motifs similar to those found in collagens and other extracellular matrix proteins, raising the possibility that the T05C12 product is a cuticle component. Consistent with this possibility, a T05C12:GFP fusion is expressed in hypodermal cells during larval and adult stages. We are currently determining the effects of expressing mutant forms of T05C12 to test the function of its Hh-C-like domain. 1. Porter, JA. et al. (1996). Cell 86, 21-34. 2. Porter, JA. et al. (1996). Science 274, 255-259. 3. Burglin, TR. (1996). Current Biology 6, 1047-1050.

Adam W Tearle et al. (2004) West Coast Worm Meeting "LEV-9 encodes a putative protein in the sarcoglycan complex"

Adam W Tearle, William R Schafer, Suchitra Jagannathan

[West Coast Worm Meeting, 2004]

The levamisole activated acetylcholine receptor in C.elegans is now known to constitute four major subunits- LEV-1, UNC-29, UNC-38 and UNC-63. Mutants for these genes are strongly resistant to the anthelminthic, levamisole. However, regulators of receptor subunit function have not all been fully characterised. Mutations in lev-9 have been shown to confer partial resistance to both levamisole and nicotine, but do not affect levamisole binding in vitro (Lewis et al ., 1987). Thus, it has been suggested that lev-9 is a possible modulator of levamisole receptor function. We have cloned lev-9 and shown that it encodes a molecule homologous to the transmembrane glycoprotein, alpha sarcoglycan. In humans, the sarcoglycans are a major component of the dystrophin glycoprotein complex (DGC) and are associated with autosomal recessive forms of limb girdle muscular dystrophy. LEV-9 is mainly expressed in the muscles of the head, pharynx, body and vulva. Using fluorescently labeled a -bungarotoxin, we have shown that lev-9 exhibits decreased surface receptor expression compared to wild type. Preliminary results using in vivo calcium imaging from vulval muscles in dissected worm preparations also suggest a difference between lev-9 and wild type in the rate of calcium influx, in response to nicotine. Further experiments using fluorescent labeling to determine how LEV-9 affects the levamisole receptor surface expression and electron microscopic comparison of lev-9 and wild types are in progress. P> P> Lewis JA, et al . (1987) J. Neurosci . 7(10), 3059-71

Anthony S Rogers (2005) International Worm Meeting "Full Length Transcripts in WormBase"

Anthony S Rogers

[International Worm Meeting, 2005]

UnTranslated Regions (UTRs) of messenger RNAs have been shown to be important in the regulation of several C.elegans genes such as tra-2(1) and fem-3(2). To give an improved representation of UTR regions in WormBase predicted full length transcripts are now generated each release. These are built up automatically from the available transcript data and the current CDS structure. Non-genomic transcript sequence such as Trans-spliced leaders and polyA tails are masked before aligning the transcript to the genome using BLAT(3). Identifying TSL and polyA sequences prior to genome alignment helps to prevent misalignments and means individual features can be made to represent these sequences. UTR structures are built from the subset of transcripts which have been computationally shown to agree with the CDS prediction. This new method of representing full length transcripts allows us to build objects that more closely resemble their biological reality with trans-spliced leader sequence and polyadenylation information included where applicable. It is also possible to show where there is evidence of multiple UTRs for a single CDS.As of WS140 there are 25869 transcripts derived from 22410 CDSs representing the 19735 protein coding genes. 1) Translational regulation of tra-2 by its 3' untranslated region controls sexual identity in C.elegans Goodwin EB, Okkema PG, Evans TC and Kimble J. Cell, 1993 75:329-339 2) Control of the sperm-oocyte switch in Caenorhabditis elegans hermaphrodites by the fem-3 3' untranslated region. Ahringer JA, Kimble JE Nature, 1991 349:346-348 3) BLAT The BLAST-Like Alignment Tool W. James Kent. Genome Research (2002) Vol. 12, Issue 4, 656-664