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[
International Worm Meeting,
2019]
Ribosome biogenesis is known to affect lifespan. The ribosome complement of the cell requires copious amounts of ribosomal RNA, which is encoded in large repetitive ribosomal DNA (rDNA) arrays in all eukaryotes. In C. elegans, N2 has 100 copies of the rDNA repeat unit, while wild isolates have ~70-400 copies. Phenotypically, severe reductions in rDNA copy number that limit ribosome biogenesis can be fatal. However, it is poorly understood how rDNA copy number variation within the range permissible for life affects phenotype. We seek to determine if differences in rDNA copy number within the range typically found in C. elegans wild isolates affect aging. To explore the potential role of rDNA copy number in aging, we have generated a set of 118 recombinant inbred lines (RILs) with high (420 copies, strain MY1) and low (130 copies, strain SEA51) rDNA copy number. We selected individuals homozygous for either 130 or 420 rDNA copies and use an automated worm imaging system (the WormBot) to determine lifespan in high-throughput. Our data will allow us to test if rDNA copy number affects lifespan, and if rDNA copy number interacts with other genetic loci. Our data indicate that the RILs show a wide range of lifespans. We are currently exploring what genetic variation contributes to this variation in lifespan. To supplement this RIL resource, we backcrossed the 130-copy rDNA array into the background of the parental 420-copy donor, and the 420-copy array into the parental 130-copy donor. These strains allow us to isolate rDNA copy number from other genetic variation. We will use this resource to test the effect of rDNA copy number variation on healthspan traits less amenable to high-throughput testing.
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[
International C. elegans Meeting,
1999]
We have tested a variety of antibodies in post-embedding protocols to localize proteins in thin sections of C. elegans. Despite recent improvements, about 50% of tested antibodies fail to work satisfactorily. Those that fail apparently involve antigens that are denatured during alcohol dehydration prior to LR Gold embedment. Fixation of chilled whole worms within a microwave oven provides more uniform, rapid fixation than worms fixed by immersion or cut open in fixative by a blade (1,2). Just as for immunofluorescence, it is still necessary to compare fixative strengths to obtain optimal preservation while retaining antigenicity. Here we compare the merits of several monoclonal and polyclonal antibodies against GFP to localize yolk protein (YP170::GFP strain) in thin sections, using a gold-linked secondary Ab. Commercial anti-GFP antibodies were obtained from Clontech, Quantum and RDI. Until now, it has been difficult to prove which organelles contain yolk, or to trace the exact path by which yolk travels from intestine to gonad and embryos (3). The YP170::GFP label has also been used by light microscopy to monitor endocytosis of yolk in wild type and mutant tissues (4). By immunoEM, GFP label in YP170::GFP animals is concentrated in two classes of dark-staining organelles in the intestine. Labeled yolk particles are found in the pseudocoelom. Labeled granules and free label above background levels are seen in the cytoplasm of oocytes in the proximal gonad and egg chamber. We will present TEM evidence for yolk passage through sheath pores and endocytosis into oocytes separately (5). We thank Barth Grant (Columbia U.) for providing the YP170::GFP strain used in this study. 1. Li and Kimble, International C. elegans Meetings, 1995, 1997 2. Miller and Hall, Worm Breeder's Gazette 15: 15, 1998 3. Bossinger and Schierenberg, Int. J. Dev. Biol. 40: 431-9, 1996. 4. Grant, Zhang, Pedraza, Hall and Hirsh, this meeting. 5. Hall, Winfrey, Blauer, Hoffman, Furuta, Rose, Hobert and Greenstein, this meeting.
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[
International Worm Meeting,
2017]
The extracellular matrix (ECM) plays an essential role in the development and function of organs and tissues. Syndecan is an ECM component that belongs to the heparan sulfate proteoglycan (HSPG) family. It is composed of 3 polysaccharidic chains linked to a core transmembrane protein. In C. elegans syndecan is coded by a single gene
sdn-1. It is required for normal vulva development and axonal guidance (Minniti et al., 2004) (Rhiner et al., 2005). Sugar chains are extensively modified including sulfation, acetylation and epimerisation of individual sugar residues. Recent reports suggest that some modifications are cell specific (Attreed et al., 2016) supporting the hypothesis of an HS code. HSPG are present at neuromuscular junctions (NMJ) but their synaptic functions remain uncharacterized. In C. elegans body-wall muscle cell receive excitatory and inhibitory innervation from cholinergic and GABAergic motoneurons, respectively. Ce-punctin / MADD-4 is an ECM protein secreted by motoneurons in the synaptic cleft. Specific combinations of Ce-punctin isoforms trigger the clustering of acetylcholine or GABAA receptors at synaptic sites. We generated a BFP knock-in allele to detect SDN-1 and observed that SDN-1 is present at NMJs and seems to be enriched at cholinergic neuro-muscular synapses. Using single-chain antibodies to label specific HS modifications in vivo suggests that some modifications could be prevalent at cholinergic junctions. Preliminary data indicate that
sdn-1 disruption affects the localization of acetylcholine receptors. Ongoing experiments and future plans will be presented at the meeting. Attreed, M., Saied-Santiago, K., Bulow, H.E., 2016. Conservation of anatomically restricted glycosaminoglycan structures in divergent nematode species. Glycobiology 26, 862-870. doi:10.1093/glycob/cww037 Minniti, A.N., Labarca, M., Hurtado, C., Brandan, E., 2004. Caenorhabditis elegans syndecan (SDN-1) is required for normal egg laying and associates with the nervous system and the vulva. J. Cell Sci. 117, 5179-5190. doi:10.1242/jcs.01394 Rhiner, C., Gysi, S., Frohli, E., Hengartner, M.O., Hajnal, A., 2005. Syndecan regulates cell migration and axon guidance in C. elegans. Development 132, 4621-4633. doi:10.1242/dev.02042
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[
International Worm Meeting,
2005]
Peptides are the original signalling molecules in metazoan nervous systems. They are also ubiquitous in nervous systems that employ classical neurotransmitters where they serve as modulators, hormones and transmitters. Understanding the role of neuropeptides is however unfortunately hindered by the absence of primary sequence information and knowledge about their post-translational modifications. This sequence information has arrived very slowly, due to the huge efforts required in tissue collection and purification to ultimately isolate and functionally characterize a peptide. In total, only 12 C. elegans peptides have been biochemical isolated and identified to date, all of which are FMRFamide-related peptides (FaRPs). The major advance of using C. elegans as a model organism in neuropeptide research is the availability of its genomic sequence [1] from which 23 FMRFamide like peptide (flp) genes [2,3] and 32 neuropeptide-like protein (nlp) genes [4] were predicted. The genomic database also represents an indispensable foundation to perform a high throughput peptidomic study. By using two-dimensional nanoscale liquid chromatography, tandem mass spectrometry and database mining, we analysed a mixed stage C. elegans extract in which we identified 28 FaRPs and 20 NLP peptides. In addition, we were able to identify 15 entirely novel peptides derived from 10 precursors that were not identified or predicted from the genome in any way previously. Some of the peptides display profound sequence similarities with neuropeptides from other animals, suggesting that they have a long evolutionary history. The present identification of novel endogenous peptides offers opportunities for a greater understanding of neuropeptide biology in C. elegans. Since this nanoscale approach worked very well for an organism as tiny as C. elegans, we are convinced that virtually any organism of which the genome has been sequenced can be analyzed by this peptidomics technology. [1] The C. elegans Sequencing Consortium (1998) Science, 282: 2012-2018. [2] Li, C, Nelson, L.S., Kim, K, Nathoo, A, Hart, A.C. (1999) Ann. N.Y. Acad. Sci., 897: 239-252. [3] Kim, K., Li, C. (2004) J. Comp. Biol., 475: 540-550. [4] Nathoo, A.N., Moeller, R.A., Westlund, B.A., Hart, A.C. (2001) PNAS, 98: 14000-14005.
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[
International Worm Meeting,
2017]
Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.
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[
West Coast Worm Meeting,
1998]
In all eukaryotes, kinesins are cytoskeletal motor proteins that mediate intracellular transport of a variety of vesicles and cellular cargo on microtubule tracks, using ATP hydrolysis. After the initial discovery of the kinesin heavy chain (KHC), a large family of kinesin like proteins has been discovered. In C. elegans using degenerate primers to the conserved ATP and microtubule binding sites, we have characterized cDNA clones corresponding to 20 genes (
klp-1 to
klp-20) encoding members of the kinesin superfamily (Khan et al., 1997). Several key questions immediately arise, such as how many different kinesins exist in one organism or cell type? Are multiple kinesins redundant or do they perform non-overlapping functions? Do other proteins exist, unrelated to the KHC, but associated with kinesins to form multimers in vivo to perform intracellular transport? In vertebrates and other higher animals, based on the structure of kinesin molecules, kinesin family members have been divided into 8 distinct classes, Group I to Group VIII (Hirokawa, 1996). Interestingly in C. elegans among the
klp-1 to
klp-20 genes, all structural types of kinesin proteins from Group I to Group VIII exist. Among these are genes that have been characterized previously such as
klp-3 (Khan et al., 1997),
osm-3 (Shakir et al., 1993; Tabish et al., 1995),
unc-104 (Otsuka et al., 1991; Hall and Hedgecock, 1991),
unc-116 (Patel et al., 1993; J. White, D. Hall, E. Hedgecock, D. Thierry-Mieg, and our unpublished data),
vab-8 (Wolf et al., 1998), and
zen-4 (Raich et al., C. elegans meeting,1997). We have begun a genetic analysis of interactions between various kinesins by constructing double mutants, gene knockouts and RNA injections to examine their phenes. RNA in situ hybridization data on whole mount embryos, using digioxiginine labeled cDNA probes from representative kinesins from all eight groups suggest that multiple kinesins co-express in early embryogenesis of C. elegans, in different cell lineages. These results provide a temporal and spatial expression pattern of the kinesin superfamily in a metazoan during early development. We thank D. Hall, N. Hirokawa, Y. Kohara, T. Motohashi, J. Miwa, K. Nisihikawa, D. T. Mieg, E. Hedgecock, R. Holmgren, and A. Otsuka, for support. Research funds were provided by the Minsitry of Education, Science, Sports and Culture, Japan and NEC Co., Japan to SSS.
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Shaevitz, Joshua, Randi, Francesco, Sharma, Anuj, Yu, Xinwei, Leifer, Andrew, Scholz, Monika, Linder, Ashley
[
International Worm Meeting,
2019]
How do patterns of neural activity across the brain represent an animal's behavior? Recent techniques for recording from large populations of neurons are providing new insights into how locomotion is encoded in population-level neural activity. Studies from mammalian systems suggest that behavioral information may be more prevalent throughout the brain and may account for a larger fraction of neural dynamics than previously thought. In C. elegans, pioneering studies revealed that the worm's neural dynamics during immobilization exhibit striking stereotyped low-dimensional patterns of neural activity that dominate brain-wide dynamics (Kato et al., 2015). These dynamics are hypothesized to map onto a motor sequence consisting of forward, backward and turning locomotion. One interpretation is that the majority of the worm brain's activity may be involved in encoding these locomotory behaviors. Here we seek to directly measure how patterns of neural activity represent locomotion by recording brain-wide calcium activity in freely-moving animals. We record calcium activity simultaneously from the majority of head neurons in C. elegans during unrestrained spontaneous locomotory behavior (Scholz et al., 2018). We find that a subset of neurons distributed throughout the head encode locomotion. By taking a linear combination of these neurons' activity, we predict the animal's velocity and body curvature and further infer the animal's posture from neural activity alone. The collective activity of these neurons outperforms single neurons at predicting velocity or body curvature. We further attempt to estimate the identity of neurons involved in the prediction. Among neurons important for the prediction are well-known locomotory neurons, as well as neurons not traditionally associated with locomotion. We compare the neural activity of the same animal during unrestrained movement and during immobilization and observe large differences in their neural dynamics. Intriguingly, during unrestrained movement we estimate that only a small fraction of the brain's overall neural dynamics are encoding velocity and body curvature. We speculate that the rest of the brain's neural dynamics may be involved in encoding other behaviors, processing sensory information or maintaining internal brain states. Kato, S., Kaplan, H.S., Schrodel, T., Skora, S., Lindsay, T.H., Yemini, E., Lockery, S., and Zimmer, M. (2015). Global brain dynamics embed the motor command sequence of Caenorhabditis elegans. Cell 163, 656-669. Scholz, M., Linder, A.N., Randi, F., Sharma, A.K., Yu, X., Shaevitz, J.W., and Leifer, A. (2018). Predicting natural behavior from whole-brain neural dynamics. BioRxiv 445643.
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[
International C. elegans Meeting,
1999]
Movement defines life. In the life of a eukaryote two major systems provide intracellular transport, using actin based myosin and microtubule based kinesin motors. The hallmark of kinesins is a 350 residue globular motor domain that contains the highly conserved ATP binding and micortubule binding sites. We have generated degenerate primers corresponding to these conserved sites and used PCR amplification to screen nematode cDNA and genomic libraries to clone and characterize cDNAs encoding more than 20 C. elegans kinesins (Khan et al., 1997; 1999). Phylogenetic tree analysis shows that the nematode kinesins can be grouped into nine distinct groups. Group-I (KHC, UNC-116); Group-II (UNC-104, KIF-1); Group-III (OSM-3, KIF3A/B); Group-IV (CEKLP-12, Chromokinesin); Group-V (CEKLP-14, DmKRP130); Group-VI (CEKLP-3; DmNcd); Group-VII (CEKLP-7, MCAK); Group-VIII (CEKLP-8, MKLP1), Group-IX (VAB-8; outgroup), representing all major classes of kinesins found in mammals. Using cellular and molecular approaches we have deduced that most of the kinesins are required for embryogenesis, such as in chromosomal movement. Post-embryonically tissue expression becomes more specific, e.g.
osm-3 is specific to a class of chemosensory neurons (Tabish et al., 1995), khc
unc-116 is expressed in motor neurons and musculature (Patel et al., 1993; Ali et al., 1999),
unc-104 is involved in synaptic vesicle transport (Hall and Hedgecock, 1991; Otsuka et al., 1991), and the
vab-8 is involved in axonal migration, and morphogenesis (Wolf et al., 1998). Analysis of the double mutants constructed with various kinesin mutant alleles reveal specific genetics interactions between nerve specific kinesins . These results allow us to propose a novel hypothesis regarding the in vivo function of different kinesins during development. which will be discussed. We thank S. Endow, L.S. B.Goldstein, G. Garriga, D. Hall, N. Hirokwa, D. T. Mieg, A. Otsuka, J. Scholey, S. Strome, J. White, for encouragement in this project. Funds were provided by Monbusho, Japan to SSS.
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[
International Worm Meeting,
2005]
The question of trans-differentiation or how a committed cell can change its identity has important implications ranging from organ regeneration to cancer. The lineage of the nematode C. elegans has identified a few cells that change their fates as the worm develops (1), and appear to be examples of trans-differentiation in vivo during wild type development. One such trans-differentiation event appears to be the transformation of Y from an epithelial cell into a motor neuron called PDA. Y is an epithelial cell that is part of the rectum in young larvae; it has the hallmarks of a polarised epithelial cell, including the presence of tight junctions (2, 3). It subsequently detaches from the rectum, moves anteriorly and becomes PDA, a motor-neuron which has a characteristic axonal projection (4, 5). Accordingly, we have determined that expression of epithelial genes - such as
ajm-1, a component of the Ce junction - is fading or lost in Y during its migration and is definitely lost in PDA. We have been characterising the steps involved in the Y-to-PDA trans-differentiation with respect to the development of the somatic gonad, and we have examined the expression of cell fate markers in both cells. We also will analyse the changes involved for these cells at the ultra-structural level, in collaboration with Dave Hall. In addition, we are determining the importance of cell to cell interactions for the Y to PDA transformation by cell ablation studies. Finally, as we are interested in understanding the molecular events underlying the Y-to-PDA cell fate transformation, we are developing a set of useful molecular markers for forward and reverse genetic screens. We will report on our progress at the meeting. 1. J. E. Sulston, H. R. Horvitz, Dev Biol 56, 110-56 (Mar, 1977). 2.
http://www.wormatlas.org. 3. J. G. White, in The Nematode Caenorhabditis elegans W. B. Wood, Ed. (Cold Spring Harbor Laboratory Press, 1988) pp. 81-122. 4. J. G. White, E. Southgate, J. N. Thomson, S. Brenner, Phil. Trans. Royal Soc. London Series B, Biol Scien. 314, 1-340 (1986). 5. D. H. Hall, R. L. Russell, Journal of Neuroscience 11, 1-22 (1991).
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[
East Coast Worm Meeting,
1998]
We have extended recently described methods for in situ patch-clamp recording from C. elegans neurons (1, 2) to permit recording in adult animals. The main improvement is the use of a fixed-stage, upright microscope mounted on an x-y translation stage. In this way, worms can be visualized from above using a high N.A. water immersion objective (Zeiss 63X/0.9 or 100X/1.0). This arrangement gives superior optics compared to viewing worms on an inverted microscope and makes it possible to expose neurons in adult animals. In addition, methods for exposing neuronal cell bodies in the head were modified to expose neuronal cell bodies in the tail for in situ patch-clamp recording. With this apparatus, we plan to record the response of PLM cells to light touch. 1. Lockery, S. R. & Goodman, M. B. (1998) Tight-seal whole-cell patch clamping of C. elegans neurons. Methods in Enzymology (in press). 2. Goodman, M. B., Hall, D. H., Avery, L. & Lockery, S. R. (1998). Active currents regulate sensitivity and dynamic range in C. elegans neurons. Neuron (in press).