Nurni Ravi, Aparna et al. (2021) International Worm Meeting "PP1/SDS-22 phosphatase is required for germ plasm segregation in the one-cell C. elegans embryo"

Araten, Alison, Miller, Stephanie, Kim, Amelia, Nurni Ravi, Aparna, Han, Bingjie, Griffin, Erik

[International Worm Meeting, 2021]

We are interested in understanding the mechanisms that control the polarization of cytoplasmic factors during asymmetric cell division. During the asymmetric division of the C. elegans zygote, the RNA-binding protein MEX-5 recruits PLK-1 kinase to the anterior cytoplasm (Nishi, 2008). In turn PLK-1 phosphorylates the RNA-binding protein POS-1, inhibiting POS-1 retention in the anterior cytoplasm (Han, 2018). As a result, POS-1 is retained and accumulates in the posterior cytoplasm, likely reflecting the activity of a phosphatase that counteracts PLK-1 phosphorylation. Through an RNAi screen to identify the phosphatase that enables POS-1 retention in the posterior, we identified the catalytic subunits (GSP-1 and GSP-2) and a regulatory subunit (SDS-22) of PP1 phosphatase. In sds-22(RNAi) embryos, MEX-5, PLK-1 and upstream PAR polarities are established normally. In contrast, POS-1 retention in the posterior cytoplasm is reduced in sds-22(RNAi) embryos, causing a weakening and delay in POS-1 segregation. Strikingly, we find that SDS-22 is similarly required for PIE-1 segregation and for P granule formation/stability, suggesting to a central role in germplasm organization. Whereas PLK-1 is enriched in the anterior, endogenously-tagged SDS-22::GFP is symmetrically distributed throughout the cytoplasm. We propose that PLK-1 kinase and PP1/SDS-22 phosphatase act in opposition to control germ plasm retention and that spatial variation in their relative activities drives the segregation of factors such as POS-1 to the posterior cytoplasm.

Rodrigues, Pedro R. et al. (2011) International Worm Meeting "Proteomic Analysis of Age-dependent Changes in Protein Solubility Identifies Genes that Modulate Lifespan."

Czerwieniec, Gregg, Gaman, Emily A., Lithgow, Gordon J., Hughes, Robert E., Rodrigues, Pedro R., Evani, Uday S., Peters, Theodore, Lucanic, Mark, Alavez, Silvestre, Mooney, Sean D., Gibson, Bradford W.

[International Worm Meeting, 2011]

While it is generally recognized that misfolding of specific proteins can cause late-onset disease states, the contribution of protein aggregation to the normal aging process is less well understood. To address this issue, a mass spectrometry-based proteomic analysis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)-insoluble conformations during aging. SDS Insoluble proteins purified from young and aged C. elegans were chemically labelled by isobaric tagging for relative and absolute quantitation (iTRAQ) and identified by liquid chromatography and mass spectrometry. Two hundred and three proteins were identified as being significantly enriched in an SDS-insoluble fraction in aged nematodes and were largely absent from a similar protein fraction in young nematodes. The SDS-insoluble fraction in aged animals contains a diverse range of proteins including a large number of ribosomal proteins. Transgenic nematodes expressing three proteins identified in the insoluble fraction, DAF-21, RPS-0 and EFT-3 fused to Green Fluorescent Protein (GFP) showed the formation of visible aggregates by fluorescence microscopy. In the case of RPS-0 and EFT-3, these aggregates appeared immobile as measured by Fluorescence Recovery after Photobleaching (FRAP). Expression of genes encoding insoluble proteins observed in aged nematodes was knocked-down in using RNAi and effects on lifespan were measured. Forty of 100 genes tested were shown to extend lifespan after RNAi. These data indicate that genes encoding proteins that become insoluble with age are modifiers of lifespan. These data also demonstrate that proteomic approaches can be used to identify genes that modify lifespan. Finally, these observations indicate that aggregation of a diverse range of proteins may be a general feature of aging.

Pu Pu et al. (2008) "Regulation of dauer formation and male tail development by a novel gene rb-13 in Caenorhabditis elegans"

Weidong Le, Ralf Baumeister, Pu Pu

[2008]

"rb-13 which was found to be upregulated in dauer larvae in a previous study, has not been functionally characterized. Firstly, by microinjection we got the expression pattern of rb-135. Secondly, we utilized rb-13 mutant and RNA interference (RNAi) to reveal the gene function of rb-13. rb-13 mutant worms showed a reduced brood size, egg-laying defect and profound male tail abnormality. Knocking down of rb-13 by RNAi also gave rise to male abnormality phenotype, similar to the phenotype observed in rb-13 mutant. The rb-13 mutant phenotype together with the RNAi results and the rb-13 spatial expression pattern confirmed that rb-13 plays an improtant role during the morphogenesis of male tail. We also found rb-13 mutant worms could form SDS-sensitive dauer like larva after starvation and these larvae are molting defect. In order to determine whether rb-13 is necessary for dauer formation, rb-13 mutant was crossed with CB1370 daf-2(e1370) which is constitutive dauer formation at 25 degree. rb-13;daf-2(e1370) double mutant worms could form constitutive dauer-like larvae but were SDS-sensitive. Since dauer is resistant to SDS, our results suggest that rb-13 is required for dauer formation and may function during the L2-to-dauer molt. "

Tohsato, Y. et al. (2019) International Worm Meeting "Data-driven analysis of female pronuclear migration by image-processing embryonic movies in Phenobank."

Kyoda, K., Okada, H., Onami, S., Tohsato, Y.

[International Worm Meeting, 2019]

Along with the recent advance of live-imaging technologies, enormous amounts of time-lapse microscopy images are becoming available in public databases. By applying image processing methods, quantitative data such as positions and shapes of nuclei and cells, and their temporal changes can be extracted from the images. Large-scale collections of such data are important resources for computational phenotype analysis. We therefore established a new resource of quantitative data on nuclear division dynamics in C. elegans RNAi-treated embryos by image-processing time-lapse DIC microscopy images in Phenobank. It consists of 1,579 sets of quantitative data from RNAi-treated embryos, including three sets of data for each of 518 genes that exerted defects in early embryogenesis when depleted individually by RNAi. Each data contains the nuclear regions and their temporal changes. The resource will be available in SSBD database (Tohsato et al. 2016; http://ssbd.qbic.riken.jp). To demonstrate the usefulness of our resource for computational phenotype screening, we calculated the speed of female pronuclear migration in its RNAi-treated embryos. 12 genes showed faster or slower migration than wild-type. Out of the 12 genes, 7 genes reproduced the migration phenotype in our independent RNAi experiments. Among the 7 genes, sds-22 and F44B9.8 exhibited remarkedly faster and slower migration respectively. sds-22(RNAi) and F44B9.8(RNAi) embryos expressing GFP::tubulin exhibited larger and smaller microtubule sperm aster respectively, consistent with the nuclear tracking along microtubules mechanism. Convergent cross mapping (CCM) identified a causal effect of the aster size on the migration speed. Since SDS-22 down-regulates ZYG-1 kinase through GSP-1/2 in centrosomal duplications (Peel et al., 2017), we examined RNAi phenotypes of gsp-1/2 in female pronuclear migration. gsp-1(RNAi) exhibited faster migration, suggesting that sds-22 regulates zyg-1 in pronuclear migrations as well. The roles of F44B9.8, an ortholog of human replication factor C, remain unclear.These results demonstrate that our resource provides new opportunities for computational phenotype screening.

Peel, Nina et al. (2013) International Worm Meeting "Centriole Copy Number Control Is Mediated by Protein Phosphatase 1-Beta in C. elegans."

O'Connell, Kevin, Iyer, Jyoti, Dougherty, Michael, Peel, Nina

[International Worm Meeting, 2013]

Centrioles are small cylindrical structures that play essential roles during cell division and development. In mitotic cells, centrioles direct formation of centrosomes, microtubule-organizing centers that promote bipolar spindle formation. In post-mitotic cells, centrioles serve as basal bodies to nucleate the formation of cilia and flagella. Under control of the kinase ZYG-1, centrioles duplicate precisely once per cell cycle with one daughter centriole assembled next to each pre-existing mother centriole. The fidelity of this process is essential for the proper segregation of chromosomes, as too few or too many centrioles lead to the formation of mono- or multipolar spindles. While defects in centriole number control have been linked to cancer and other diseases, the mechanisms that constrain centriole assembly to one daughter per mother are poorly understood. We have found that loss-of-function mutations that affect either of two highly conserved PP1 regulators (referred to here as I-2 and SDS-22) can suppress the centriole duplication defect incurred by partial loss of ZYG-1 activity. Likewise, reduced expression of GSP-1, the worm PP1-beta catalytic subunit also suppresses a zyg-1 mutant. Proteomic analysis indicates that both I-2 and SDS-22 bind GSP-1, but function in distinct PP1 complexes. Significantly, we find that loss of PP1-beta activity leads to the over-expression of ZYG-1, suggesting a likely mechanism of suppression. Most interestingly, in a zyg-1(+) background, strong inhibition of I-2 or SDS-22 activity leads to centriole over-duplication whereby mother centrioles produce more than a single daughter. Our results show that by limiting ZYG-1 expression, PP1-beta acts as a key determinant of centriole number.

Nika, Liberta et al. (2015) International Worm Meeting "Fluorescent beads are a versatile tool to distinguish dauer larvae and other stages."

Nika, Liberta, Konkus, Rebecca, Gibson, Taylor, Karp, Xantha

[International Worm Meeting, 2015]

C. elegans life history is dependent on environmental cues transduced through several signaling pathways. In favorable environmental conditions C. elegans develops continuously through four larval stages before molting into the adult stage. In contrast, adverse environmental conditions promote entry into the stress resistant dauer larva stage immediately following the second larval molt. Dauer larvae possess distinct properties including cellular and developmental arrest, extended lifespan, and discontinued feeding. If environmental conditions improve, dauer larvae recover to post-dauer L3 larvae, which are developmentally identical to continuously developing L3 larvae. Studies of dauer larvae have seeded discoveries in diverse fields, ranging from aging to neurobiology. Many of these studies rely on the unambiguous identification of dauer larvae. The most useful method to identify dauer larvae would be simple, scalable, and feasible even in mutant backgrounds that are defective in some aspects of dauer morphogenesis. None of the current methods possess all of these qualities. Here, we describe such a method taking advantage of the inability of dauer larvae to feed. Fluorescent beads are added to the bacterial food source, and dauer larvae are identified by a lack of beads within their digestive tract. We describe how this assay can be used to isolate dauer larvae formed by any of three common methods: starved plates, exogenous pheromone, and dauer-constitutive mutations. Lack of beads correlates well with other known markers of dauer formation, including greatly reduced pumping rate, presence of dauer alae and SDS resistance. We find that using beads rather than SDS-resistance to identify dauer larvae enables the recovery of SDS-sensitive mutants, including cuticle mutants and certain mutants within the dauer formation pathway. Finally, lack of beads also identifies molting larvae, extending the utility of this assay beyond dauer larvae.

Rozemuller, M. et al. (2019) International Worm Meeting "Individuality in C. elegans turning behaviour affects its motile efficiency."

Rozemuller, M., Shimizu, T.

[International Worm Meeting, 2019]

To survive, organisms continuously make navigational decisions based on limited available information. Even in isogenic populations difference in internal state and experience can result in different phenotypes. We explore reorientation statistics of C. elegans performing three contrasting tasks: (i) roaming for food, (ii) escaping from an SDS stimulus, and (iii) chemotaxis up a salt gradient, and ask how variability impacts its efficiency. In the absence of a stimulus we find that worm crawling demonstrates directional bias, which fluctuates on a ~1 hr time scale . Our results suggest that a strong crawling bias impairs roaming and chemotactic efficiency. Significant worm-to-worm variability (individuality) is observed for the frequency of sharp reorientations (?-turns). An increased sharp-turn rate is associated with less efficient roaming and chemotaxis. Minimalistic modeling of the worm's trajectory statistics confirms that increased crawling bias and sharp-turn frequency reduces roaming efficiency. Interestingly the model predicts that an increased sharp-turn frequency can be beneficial for worms with a strong crawling bias. In addition, during sharp reorientations worms also show individuality in dorso-ventral preference and amplitude, which is not associated with increased roaming efficiency. However, we do find that during SDS avoidance, both the sharp turn turning direction and amplitude are modulated in a meaningful way to minimize the exposure. Our results provide evidence that individual differences and temporal modulations of turning statistics affect the efficiency of motility tasks such as exploration, exploitation and avoidance.

Andersen*, Kristine et al. (2015) International Worm Meeting "An Investigation into the Affect of Neuronal Activity on Proper Neural Connectivity in C. elegans."

Andersen*, Kristine, Varshney, Aruna, Duong, Alex, Miller, Kristine, Park, Joori, Pyle, Jacqueline, Barsi-Rhyne*, Benjamin, VanHoven, Miri, Vargas, Christopher, Holdrich, Emma, Tsujimoto, Bryan, Tran, Alan

[International Worm Meeting, 2015]

Neuronal activity has been implicated in the establishment and maintenance of appropriate synaptic connections in vertebrate and invertebrate systems. However, the molecular mechanisms by which neuronal activity affects connectivity are poorly understood. To understand this process, we have focused our studies on the PHB phasmid sensory neurons. We have begun to elucidate the pathway by which sodium dodecyl sulfate (SDS) is sensed by the phasmid neurons using a high throughput assay adapted from the method developed by Hilliard and colleagues (Current Biology, 2002). Briefly, SDS is sensed by the PHB neurons, which terminate backward movement via their connections with AVA interneurons. Using this assay, we find that odr-3/Galphaolf and tax-2/CNG-channel beta subunit, in addition to previously discovered tax-4/CNG-channel alpha subunit (Hilliard et al., Current Biology, 2002), are required for SDS chemosensation. To determine if defects in sensory signaling affect sensory synapses, we utilized the split-GFP-based trans-synaptic marker NLG-1 GRASP (Neuroligin-1 GFP Reconstitution Across Synaptic Partners) to visualize synapses between PHBs and AVA interneurons in live animals. Interestingly, we find that neuronal activity is required for maintaining these sensory synapses. Time course experiments indicate that odr-3/Galphaolf mutant L1s have normal synapses, but synapses are significantly reduced in later larval stages, although phasmid neuron morphology appears to be unaffected. Cell-specific rescue experiments indicate that odr-3/Galphaolf likely functions in PHB neurons. Subcellular localization of odr-3/Galphaolf shows localization to the PHB cilia, consistent with a role in sensory signaling being required to maintain synaptic integrity. These results indicate that C. elegans may be a powerful model organism for elucidating the molecular mechanisms by which sensory activity affects synaptic connectivity. Our future goal is to further characterize the mechanism by which sensory activity maintains synapses using molecular genetic and physiological approaches. Funded by NIH (1R01NS087544 to MV at SJSU and NL at UCSF, 5T34GM008253 MARC undergraduate fellowship to CV, 2R25GM071381 RISE undergraduate fellowships to CV and JP), HHMI (SCRIBE 52006312 undergraduate fellowship to BB and KM), and NSF (RUMBA REU 1004350 fellowship to KA and EH).

Huang X-Y et al. (1991) International C. elegans Meeting "BIOCHEMICAL CHARACTERIZATION OF TWO PROTEINS SPECIFICALLY ASSOCIATED WITH THE TRANS-SPLICED LEADER"

Hirsh DI, Huang X-Y

[International C. elegans Meeting, 1991]

RNA trans-splicing in nematodes involves the transfer of an evolutionarily conserved 22-nucleotide spliced-leader (SL) and a trimethylguanosine (TMG) cap to the 5'-end of the recipient mRNA. The physiological role of trans-splicing, of the SL and the biological effect of TMG cap at the 5' ends of mRNAs are not yet understood. The possibility has been raised that SL itself has a catalytic role in splicing. The TMG cap has recently been shown to be an essential nuclear targeting signal. We hope to learn the functions of SL or transsplicing by identifying proteins specifically bound to SL and elucidating their functions. Electrophoretic mobility shift assays combined with competition analysis showed two proteins SLBP1 and SLPB2, bind specifically to SL1. The binding can be stimulated by a cap structure at the 5' end of SL. Although SLBP2 can bind SL1 RNA, SLBP1 seems unable to bind SL1 RNA. This may be due to the highly structured SL1 RNA interfering SLBP1 binding. We are presently testing the binding of SLBP1 and SLBP2 to SL2. UV cross-linking and SDS-PAGE revealed that SLBP1 has a molecular mass of 57kD and SLBP2 of 30kD. Nitrocellulose filter retention assay showed that the binding between SLBP1 and SL1, SLBP2 and SL1 conforms to a simple biomolecular reaction. SLBP1 has been purified to homogeneity as judged by silver staining on SDS-PAGE. Gel filtration analysis indicates that SLBP1 exists as a monomer and a dimer in solution. However we do not know whether dimerization is required for RNA binding. We intend to isolate their genes and generate antibodies against SLBP1 and SLBP2 to further characterize their biochemical properties and biological functions.

Kelley L Banfield et al. (2003) International Worm Meeting "Interplay of the pcm-1 methyltransferase and the daf-2 receptor kinase in dauer larvae formation and longevity"

Pamela L Larsen, Kelley L Banfield, Steven Clarke

[International Worm Meeting, 2003]

At least one type of spontaneous protein damage that develops during the aging process can be recognized and partially repaired by the protein L-isoaspartyl-O-methyltransferase. This enzyme is encoded by the pcm-1 gene in Caenorhabditis elegans. Deletion of pcm-1 has no effect on brood size or embryonic lethality. In addition, deletion of pcm-1 has no effect on adult life span even in the presence of a mutation in the daf-2 gene that doubles adult life span. However, the pcm-1 deletion does affect the formation and longevity of dauer larvae. In response to a pheromone signal, animals lacking pcm-1 inefficiently form a mixture of dauer and dauer-like larvae. Of these, only 20% are properly formed dauer larvae as determined by their resistance to sodium dodecyl sulfate (SDS). The SDS-sensitivity of the pcm-1 mutants is suppressed by the daf-2(m41) mutation in the insulin receptor-like tyrosine kinase gene which causes constitutive dauer larvae formation. The small fraction of properly formed pcm-1 dauer larvae shows reduced survival at 20o with a 50% survival of less than 5 days compared with approximately 40 days for the N2 wild-type strain. Life span of daf-c mutant dauer larvae is extended compared to wild type at 20o, with a 50% survival of about 55 days, for the daf-7 mutant, the daf-2 mutant, and the daf-2; pcm-1 double mutant. Interestingly, the survival of dauer larvae from all genotypes, except the pcm-1 mutant, is decreased for dauer larvae cultured at 25o compared to 20o, with a 50% survival of about only 25 days for the N2 wild-type strain and only 35 days for the daf-7, daf-2, and daf-2; pcm-1 strains. The reduced survival at 25o compared to 20o of this 'non-aging' larval stage resembles that previously observed for adults. The reduced ability of pcm-1 mutants to properly form dauer larvae, as well as the poor survival of the small fraction of SDS-resistant pcm-1 deficient dauer larvae, indicate an important role for the PCM-1 methyltransferase in forming and maintaining the dauer stage. It is unclear whether the growth signal is strengthened or the dauer larva-inducing signal is compromised in the absence of PCM-1. Moreover, proper signal integration during the dauer entry process seems to affect the longevity of the stage. The fact that morphologically-similar dauer larvae differ in their survival suggests that physiological differences exist between genotypes and that efficient life maintenance in this stage is malleable, as is the case in adult worms.