Chonglin Yang et al. (2003) International Worm Meeting "Modulation of Programmed Cell Death with RNA Aptamers"

Xiaochen Wang, Ding Xue, Jay Parrish, Chonglin Yang

[International Worm Meeting, 2003]

Programmed cell death (apoptosis) plays an essential role in maintaining the physiological balance of appropriate cell numbers by opposing uncontrolled cell proliferation. The pathway of programmed cell death appears to be highly conserved from C. elegans to mammals, suggesting that studies of programmed cell death in C. elegans can provide important information for understanding how cell death is regulated and executed in mammals. Moreover, novel ways developed in C. elegans to modulate programmed cell death may also be applied to mammals for better detection, prevention as well as treatment of human diseases caused by abnormal apoptosis (e.g. cancer, autoimmune disorders, and neurodegenerative diseases). In this study, we are employing the technique of SELEX (Systematic evolution of ligands by exponential amplification) to identify small RNA aptamers with high binding specificity and affinity for key cell death regulators, including CED-9 and CED-4 from C.elegans as well as Bcl-2 and Bcl-xL from humans. We hope to use these RNA aptamers to probe how Bcl-2 family proteins regulate programmed cell death in both C. elegans and mammalian cells. More importantly, if these RNA aptamers can be used to successfully modulate the process of programmed cell death in C. elegans, they may provide important insights into devising new diagnostic and therapeutic drugs to treat cancer and various apoptosis-related diseases. So far, we have conducted several rounds of SELEX experiments on CED-9, CED-4 and Bcl-xL and have obtained candidate molecules with increasing binding affinity in vitro to these proteins. After more rounds of selections, we will investigate in detail the potential effects of candidate RNA aptamers on in vitro activities of these key cell death regulators as well as their potential effects on apoptosis in C. elegans and in mammalian cells.

V Kapulkin et al. (2001) International C. elegans Meeting "The worm is not enough - comparative genomics of Ancylostoma ceylanicum"

A Ciesielski, A Magalska, H Wedrychowicz, V Kapulkin

[International C. elegans Meeting, 2001]

Ancylostoma ceylanicum is a parasitic nematode which infects humans and domesticated animals. We have taken a comparative genomic approach to understanding A. ceylanicum parasitism by constructing cDNA and genomic DNA libraries from this parasitic nematode. We build cDNA libraries using directional, SL1- oligo dT, RT-PCR amplification, followed by topoisomerase I- based A/T cloning into a pCR-XL-TOPO (Invitrogen). PCR products were size selected by horizontal gel slicing and fractions were subsequenty separately cloned to build sub- libraries. In our view this strategy allows rapidly identification of highly complex sub- libraries and therefore eliminate fractions containing highly redundant inserts. Using similar strategy we build a small insert genomic library. Briefly, genomic DNA was sheared, gel fractionated and cloned into pCR4Blunt-TOPO (Invitrogen). Both cDNA and gDNA libraries were therefore build in high-copy plasmid based vectors useful for direct sequencing. To characterize our libraries we sequenced over one hundred clones. We find that about half of SL-cDNA clones show apparent homology to genes predicted by C. elegans genome project. Results of trial sequencing and characteristics of sub- libraries will be summarized.

Hersh BM et al. (1997) International C. elegans Meeting "SCREENING FOR SUPPRESSORS OF ced-9(n1950)"

Horvitz HR, Hersh BM

[International C. elegans Meeting, 1997]

The C. elegans cell-death gene ced-9 is a member of the Bcl-2 family of cell-death regulators. Some mammalian members of this family, such as Bcl-2 and Bcl-xL, protect against cell death, while others, such as Bax and Bak, promote cell death. However, the mechanisms by which Bcl-2 family members exert their effects remain unclear. ced-9 appears to be a protective member of the family, as loss-of-function mutations in ced-9 result in ectopic cell death and maternal-effect lethality, while overexpression of ced-9 blocks programmed cell death. The ced-9(n1950) allele, which causes a glycine-to-glutamate substitution in the conserved BH1 domain, acts as a gain-of-function mutation in C. elegans, preventing all programmed cell deaths during development. To identify genes that may interact with ced-9 or that may be necessary for the protective effect of the ced-9(n1950) allele, we have begun screening for suppressors of the Ced phenotype of ced-9(n1950) animals. We mutagenized sem-4(n1378) ced-1(e1735); ced-9(n1950) animals and looked for the restoration of cell corpses in either F2 or F3 embryos. We have screened approximately 10,000 haploid genomes for zygotic suppressors, and 1,500 haploid genomes for maternal-effect suppressors. We have isolated two candidates from the maternal-effect screen, both of which are maternal-effect lethal. We are currently mapping these candidates and testing them for complementation with ced-9 loss-of-function alleles. In addition, we have generated anti-CED-9 antibodies and are currently characterizing the expression pattern of CED-9 protein in both wild-type and mutant backgrounds.

Hersh BM et al. (1996) East Coast Worm Meeting "STRUCTURE-FUNCTION ANALYSIS OF CED-9"

Horvitz HR, Hersh BM

[East Coast Worm Meeting, 1996]

The C. elegans cell-death gene ced-9 is a member of the Bcl-2 family of cell-death regulators. Some members of this family, such as Bcl-2 and Bcl-xL, protect against cell death, while others, such as Bax and Bak, promote cell death. However, the mechanism by which these family members exert their effects remains unclear. ced-9 appears to be a protective member of the family, as loss-of-function mutations in ced-9 result in ectopic cell death and maternal-effect lethality, and overexpression of ced-9 blocks programmed cell death. We have begun a structure-function analysis to determine which portions of the CED-9 protein are necessary for its protective effect in vivo. Members of the Bcl-2 family are most similar in three domains, BH1, BH2, and BH3. We generated and assayed for function both deletion constructs and site-specific mutations in the conserved BH1 and BH2 domains of CED-9. Constructs were placed under the control of the heat-shock promoters and tested for the ability to block programmed cell death, as measured by the presence of extra cells in the anterior pharynx. Constructs were also tested for the ability to rescue ced-9(lf) mutants. Mutation of the conserved BH1 residue glycine-169 to alanine reduced, but did not completely eliminate, the protective ability of CED-9. This construct was still able to rescue ced-9(lf) in 1/5 transgenic lines. An identical change in Bcl-2 eliminates both protective function and the ability of Bcl-2 to heterodimerize with Bax. This residue is mutated to glutamate in the ced-9(n1950) gain-of-function mutant. Mutation of the conserved BH1 residue phenylalanine-177 to alanine did not affect the protective effect of ced-9 overexpression, and this construct was able to rescue ced-9(lf) in 4/5 transgenic lines. Thus, even residues that are conserved across all Bcl-2 family members may not be essential for the protective function of CED-9. Overexpression of a deletion construct removing residues 78-192, thereby eliminating both the BH1 and BH3 domains but leaving the BH2 domain intact, did not block programmed cell death. We intend to generate more deletion constructs and site-specific mutations to continue our analysis.

Mirzakhalili, Ehsan et al. (2017) International Worm Meeting "A computational model of the calcium dynamics in Caenorhabditis elegans ASH sensory neuron."

Gourgou, Eleni, Epureanu, Bogdan, Mirzakhalili, Ehsan

[International Worm Meeting, 2017]

C. elegans is widely used as a model system for monitoring stimulus-evoked Ca2+ transients in neurons. The ASH sensory neuron is the subject of several such studies, primarily due to its key importance as a polymodal nociceptor. However, despite the pivotal role of ASH in C. elegans, the overall biology and the characteristics of its Ca2+ transients (e.g., the "off" response), no mathematical model has been developed to describe the full mechanism of ASH Ca2+ dynamics. We propose a phenomenological computational model which captures the Ca2+ transients in the C. elegans ASH neuron upon its activation. The model is built on biophysical cascades that unfold as part of the neuron's Ca2+ signaling events and homeostatic mechanism (e.g., TRPV channels and voltage-gated channels activation, Ca2+ release from intracellular stores, IP3 dynamics, PMCA and SERCA pumps function). The state of the ion channels is described based on Hodgkin-Huxley equations and the remaining molecular states are based on kinetic equations with phenomenological adjustments. We fit the model using experimental data of osmotic stimulus-evoked ASH Ca2+ transients, detected with a FRET sensor (TN-XL), in young and aged worms, both untreated and exposed to oxidative stress. We use a multi-objective genetic algorithm to find the parameters for young untreated worms' data set. Parameters are estimated using a hybrid method that consists of a genetic algorithm and nonlinear least-squares. We use the same approach to fit the model for the other groups of experimental data. We validate the model using data from the literature, from ASH activation by stimuli of several strengths and durations. Finally, we demonstrate how our model can be used to predict the ASH Ca2+ response to stimulation pulses that are challenging to achieve experimentally (stimuli sequences of varying durations/lengths, or ramp stimuli). Our model includes for the first time the changes in ASH cytoplasmic Ca2+ flux observed both upon delivery and withdrawal of the stimulus (i.e., the "on" and "off" responses). This effort is the first to propose a quantitative dynamic model of the Ca2+ transients generating mechanism in a C. elegans neuron, based on essential biochemical pathways of the Ca2+ homeostasis machinery.

Jessica Tanis et al. (2001) International C. elegans Meeting "unc-20 MAPS TO A REGION THAT INCLUDES TWO PREDICTED GOLGI COMPLEX PROTEINS"

Peter Alf, Brook Kohrt, Jeff Doto, Jessica Tanis, Bruce Wightman

[International C. elegans Meeting, 2001]

The canonical weak unc-20 mutation, e112 , causes a characteristic coiling uncoordinated defect, as well as defects in axon outgrowth and pathfinding in several neurons, and increased sensitivity to serotonin. Strong unc-20 mutations, recovered as part of a smg- dependent screen performed by Phil Anderson's lab, cause developmental arrest in late embryogenesis or at hatching. Given the pleiotropies of unc-20 mutations, the gene may be involved in a central cellular process, either one specific to the nervous system or a general process. Genetic mapping of unc-20 places it between imo-1 and fax-1 on XL. This region is covered by genomic cosmid clone C15C7, however we have been unable to obtain stable transformants with this clone, preventing us from determining the location of unc-20 by transformation rescue experiments. Cosmids to the left and right of C15C7 do not rescue unc-20 and a large deletion just to the right of C15C7 complements unc-20 . Therefore, we have focused our attention on the predicted genes on cosmid C15C7. We amplified genomic DNA from unc-20(e112) and unc-20(r977) homozygous animals from regions of C15C7 that correspond to predicted genes C15C7.3, C15C7.4 and C15C7.5; all sequences were the same as wild-type. RNAi experiments with a cDNA clone corresponding to C15C7.5 (yk64b6; obtained from Y. Kohara) did not generate any obvious visible phenotype. Therefore, we conclude that these three genes are unlikely to encode unc-20. The two remaining candidates, C15C7.1 and C15C7.2, appear to be transcribed together in an operon. Both predicted genes encode products that are predicted to be localized to trans-Golgi. C15C7.1 encodes a protein that is similar to syntaxin-6, a SNARE that is thought to function in trans - Golgi vesicle trafficking. C15C7.2 encodes a protein that includes a coiled-coil region and a C-terminal GRIP domain. The GRIP domain is associated with vertebrate proteins that are localized to trans-Golgi. One can imagine mechanisms by which defects in vesicle trafficking and/or secretion may result in all known unc-20 phenotypes. For example, axon pathfinding and extension defects may be the result of difficulties in supplying new membrane to growth cones or the result of compromised transport of receptor molecules. RNAi experiments with a cDNA clone corresponding to C15C7.2 (yk121h6) causes weak embryonic lethality, but does not cause the Unc phenotype characteristic of unc-20 putative hypomorphs. We are currently amplifying DNA corresponding to both of these predicted genes from unc-20 mutants; come to our poster for the outcome. We are also embarking on an electron microscopic study of unc-20 mutants. We will evaluate global nervous system phenotypes, such as integrity of axon bundles, as well as subcellular Golgi and vesicle anatomy.

Greenstein D et al. (1991) International C. elegans Meeting "THE POU GENE CEH-18 IS EXPRESSED IN MANY DIFFERENT CELL TYPES"

Finney M, Greenstein D, Ruvkun GB, Hird SN

[International C. elegans Meeting, 1991]

The POU domain is a protein motif consisting of a specialized homeodomain and an adjacent region of extended sequence similarity first found to be shared among the mammalian transcription factors Pit- 1 and Oct-1 and the C. elegans cell lineage control gene unc-86. The POU domain directs cooperative binding to specific DNA sequences. We decided to clone additional members of the POU family from C. elegans and to undertake a combined molecular and genetic analysis of their function. We hope to elucidate the various roles that POU genes might play in specifying cell fate and directing the C. elegans cell lineage. We have focused on one such gene, ceh-18, which has been analyzed by in situ hybridization and antibody staining. In contrast to unc-86, which is only expressed in neuroblasts and neurons, ceh-18 is expressed in a variety of different cell types, though is notably absent from nearly all neurons. ceh-18 was isolated by screening Stuart Kim's cDNA library with degenerate oligonucleotide probes designed to hybridize to POU domain genes. Subsequently, genomic clones were isolated, placing ceh-18 on XL. DNA sequence analysis of a 2.2 kb cDNA indicates that ceh-18 contains a POU domain that defines a new class of POU proteins. We studied the spatial distribution of ceh- 18 transcripts in embryos by in situ hybridization to whole-mount preparations using digoxigenin-labeled probes and fluorescent detection. ceh-18 mRNA is first detected at the three-fold stage of embryogenesis and is most evident in pharyngeal muscle cells, hypodermal cells of the head and tail, and what appear to be body muscle cells. We raised a polyclonal antiserum to the ceh-18 protein to study its expression further. The antiserum detects a 72 kdal and a 65 kdal protein in Western blots, with each protein being present at all stages of development. In immunofluorescence experiments, the affinity purified antiserum stains nuclei of a variety of differentiated and blast cells. Since we do not yet have a ceh-18 protein null mutant, we cannot be sure that all of the immunoreactivity is ceh-18 protein. However, in agreement with the in situ hybridization experiments, the antibody stains the nuclei of the pharyngeal muscle cells, hypodermal cells, and body muscle cells at the three-fold stage of embryogenesis. ceh-18 immunoreactivity is also found in embryonic nuclei even prior to the first division. The pattern of ceh-18 expression is dynamic with a variety of different cell types expressing the protein depending on developmental stage. Other examples of staining cells include the nuclei of hypodermal cells, body muscle cells, intestinal cells, and the distal tip cells. So far the only neurons seen to strongly express the ceh-18 protein are ALML/R. We are pursuing a number of strategies to obtain ceh-18 mutations.

Hersh BM et al. (1998) East Coast Worm Meeting "ced(n3194), A SUPPRESSOR OF ced-9(n1950), MAY REPRESENT A NEW NEGATIVE REGULATOR OF CELL DEATH"

Hersh BM, Horvitz HR

[East Coast Worm Meeting, 1998]

The C. elegans cell-death gene ced-9 is a member of the Bcl-2 family of cell-death regulators. Some mammalian members of this family, such as Bcl-2 and Bcl-xL, protect against cell death, while others, such as Bax and Bak, promote cell death. ced-9 appears to be a protective member of the family, as loss-of-function mutations in ced-9 result in ectopic cell death and maternal-effect lethality, while overexpression of ced-9 blocks programmed cell death. The ced-9(n1950) allele, which causes a glycine-to-glutamate substitution in the conserved BH1 domain, acts as a gain-of-function mutation in C. elegans, preventing all programmed cell deaths during development. To identify genes that may interact with ced-9 or that may be necessary for the protective effect of the ced-9(n1950) allele, we screened for suppressors of the Ced phenotype of ced-9(n1950) animals by looking for the appearance of corpses in embryos. From a screen of 4,500 haploid genomes for maternal-effect suppressors, we isolated one candidate, n3194, with a large number of refractile objects in embryos. n3194 maps to LGIII, complements ced-9(lf), and has a recessive maternal-effect lethal phenotype. Neither the accumulation of refractile objects nor the lethality was suppressed by loss-of-function mutations in ced-3 or ced-4. This result suggests that the gene defined by n3194 either acts downstream of or in parallel to ced-3 and ced-4. Either n3194 defines a new gene involved in programmed cell death or the refractile objects are not programmed cell deaths. We are attempting to use acridine orange staining and TUNEL staining to determine if the refractile objects observed in n3194 mutants correspond to corpses generated by programmed cell death. In addition, we have constructed double mutants with ced-1 and ced-8, which affect the engulfment of corpses and the timing of the appearance of cell corpses, respectively. These double mutants will be analyzed to determine if the refractile objects behave genetically as corpses. We intend to use electron microscopy to further analyze cell-corpse morphology in the n3194 mutant to determine if the refractile objects are corpses. We mapped n3194 and obtained transformation rescue. We have narrowed the rescuing region to an 8.5 kb fragment containing a single predicted gene. The predicted protein is similar to the products of a number of human and mouse ESTs of unknown function. ced(n3194) may represent a new negative regulator of cell death. If so, suppressors of n3194 lethality may help identify new components of a cell-death pathway. In a pilot screen of 600 haploid genomes, we have identified two candidate suppressors of the lethal phenotype.