Chun-Yi Huang et al. (2007) International Worm Meeting "eif-3.K is a pro-apoptotic gene in C. elegans."

Jia-Yun Chen, Yi-Chun Wu, Chun-Yi Huang

[International Worm Meeting, 2007]

Programmed cell death (or apoptosis) is an important feature of C. elegans development. Previous studies have identified pro-apoptotic genes egl-1, ced-3 and ced-4 and anti-apoptotic genes ced-9 and icd-1 that control programmed cell death.. We have identified and characterized a novel pro-apoptotic gene eif-3.K. Loss-of-function by mutation or RNAi inactivation in eif-3.K resulted in a decrease of cell corpses, whereas heatshock-induced over-expression of eif-3.K weakly but significantly increased cell corpses. Interestingly, the eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of egl-1 or ced-4. This result suggests that eif-3.K may act downstream of or in parallel to egl-1 and ced-4 in the programmed cell death pathway. Using a cell-specific promoter to express eif-3.k in touch neurons, we showed that eif-3.K likely promoted cell death in a cell-autonomous manner. To further explore EIF-3.K function, we generated antibodies against bacterially expressed EIF-3.K protein. We found that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. As human eif-3.K can functionally substitute C. elegans eif-3.K in an eif-3.K mutant, the function of eif-3.K in apoptosis is likely conserved in evolution.

Chun-Yi Huang et al. (2006) East Asia C. elegans Meeting "eif-3.K is a pro-apoptotic gene in C. elegans"

Teng-Wei Huang, Chun-Yi Huang

[East Asia C. elegans Meeting, 2006]

Programmed cell death or apoptosis is an important feature during C. elegans development. The pro-apoptotic genes egl-1, ced-4 and ced-3 are required for the execution of cell death. We have identified and characterized a novel pro-apoptotic gene eif-3.K. A loss-of-function mutation or inactivation by RNA interference in eif-3.K resulted in a reduction of cell corpse number during embryogenesis, whereas heatshock-induced over-expression of eif-3.K weakly but significantly promoted programmed cell death. In addition, eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of egl-1 and ced-4. This result suggests that eif-3.K may act downstream of or in parallel to egl-1 and ced-4 in the genetic pathway during programmed cell death. Using a cell-specific promoter we showed that eif-3.K likely promoted cell death in a cell-autonomous manner. We generated antibodies against bacterially expressed EIF-3.K protein. The immunostaining result showed that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. To better understand the cell-death defect of eif-3.K mutants, we are currently performing a 4D microscopic analysis of the cell death process in wild-type and eif-3.K mutants.

Anita Fernandez et al. (2006) Development & Evolution Meeting "A Network Model of C. Elegans Early Embryogenesis Identifies a Novel AT-Hook Protein that Coordinates Nuclear Envelope Function and Chromatin Maintenance"

Anita Fernandez, Fabio Piano

[Development & Evolution Meeting, 2006]

Using combined network analysis of large-scale functional genomic data we mapped multi-protein modules required for distinct processes during early embryogenesis (Gunsalus et al. 2005). A basic question is how these molecular modules are coordinated through the mitotic cycle to ensure the proper unfolding of early developmental events. To identify proteins that could coordinate different modules we searched for proteins that bridged different modules. One such protein, C38D4.3, could be placed in either the nuclear pore complex module or in the chromosome maintenance module by a network clustering algorithm M-CODE (Bader et al. 2003). Consistent with its predicted roles at the nuclear pore and in chromosome segregation, GFP fusions and anti-C38D4.3 immmunolocalizations show that C38D4.3 shuttles between the nuclear envelope and the kinetochore during the cell cycle. Functionally, C38D4.3 is required for proper nuclear envelope and chromatin maintenance. C38D4.3 (RNAi) embryos, like embryos without nuclear pore components, are incapable of completely separating cytoplasm from nucleoplasm, failing to exclude microtubules and affecting the nuclear localization of PIE-1, a protein normally enriched in the P1 nucleus (Mello, 1996). Additionally, pronuclei fail to meet, and centrosomes do not remain attached to the paternal pronucleus and segregate prematurely. In these embryos, metaphase spindles are not established and chromatin neither condenses, congresses, nor segregates properly. These phenotypes are reminiscent of RNAi phenotypes of genes from the Ran GTPase cycle (Askjaer 2002). Looking for C38D4.3 (RNAi) phenotypic neighbors using PhenoBlast (Gunsalus et al 2004) or phenoclusters from large-scale RNAi analyses (Sonnichsen et al 2005; Gunsalus et al 2005) we identified ~25 other genes with similar defects when analyzed by time-lapse Nomarski microscopy. Of these, genes that are part of the RanGTPase pathway (ran-1, ran-2, and npp-9) were required for proper C38D4.3 localization. Thus C38D4.3 is critical for both mitotic and interphase cell functions and is a likely target of the Ran GTPase pathway.

Johnson, Christina K. et al. (2019) International Worm Meeting "Requirement of glial Na+/K+-ATPase in touch sensation highlights ionic and metabolic link between glia and touch neurons in C. elegans."

Johnson, Christina K., Bianchi, Laura, Wang, Ying, Fernandez Abascal, Jesus

[International Worm Meeting, 2019]

Isolated microenvironments, such as the tripartite synapse, where the concentration of ions is regulated independently from the surrounding tissues, exist throughout the nervous system, including in mechanoreceptors. Modulation of the ionic composition of these microenvironments has been suggested to be achieved by glia and other accessory cells. However, the molecular mechanisms of ionic regulation and effects on neuronal output and animal behavior are poorly understood. Using the model organism C. elegans, our lab published that Na+ channels of the DEG/ENaC family expressed in glia control neuronal Ca2+ transients and animal behavior in response to sensory stimuli. DEG/ENaC Na+ channels are known to establish a favorable driving force for K+ excretion, which occurs via inward rectifier K+ channels, in epithelial tissues across species. We hypothesized that a similar mechanism exists in the nervous system. Using molecular, genetic, in vivo imaging, and behavioral approaches, we showed that expression in glia of inward rectifier K+ channels and cationic channels rescues the sensory deficits caused by knock-out of glial DEG/ENaCs without disrupting neuronal morphology, supporting our hypothesis. Based on this model, Na+/K+-ATPases are also needed to maintain ionic concentrations following influx of Na+ and excretion of K+. We show here that, in addition to glial Na+ and K+ channels, two specific glial Na+/K+-ATPases, EAT-6 and CATP-1, are needed for touch sensation and that their requirement can be bypassed by a high glucose diet. The effect of glucose is dependent on ATP binding capability of the pump, translation, transcription, and the activity of CATP-2, a third Na+/K+-ATPase ?-subunit. Taken together, our results support metabolic and ionic cooperation between glia and neurons in C. elegans mechanosensors, a mechanism that is essential to regulating neuronal output and may be conserved across species.

Boxem, Mike (2009) International Worm Meeting "Mapping the protein interaction network that controls cell polarity."

Boxem, Mike

[International Worm Meeting, 2009]

Interactions between proteins are a key component of most or all biological processes. A key challenge in biology is to generate comprehensive and accurate maps (interactomes) of all possible protein interactions in an organism. This will require iterative rounds of interaction mapping using complementary technologies, as well as technological improvements to the approaches used. For example, we recently developed a novel yeast two-hybrid approach that adds a new level of detail to interaction maps by defining interaction domains(1). Currently, I am working to generate an interaction map of proteins involved in controlling cell polarity in C. elegans to improve our understanding of the molecular mechanisms that establish and maintain cell polarity in multicellular organisms. I will combine two fundamentally different interaction mapping techniques: the yeast two-hybrid system (Y2H) and affinity purification/mass spectrometry (AP/MS). This will provide more detail by identifying both direct interactions between pairs of proteins by Y2H, and the composition of protein complexes by AP/MS. Moreover, interactions missed by one technology may be detected by the other, leading to a more complete interaction map. I will integrate the physical interactions with phenotypic characterizations. To this end I will systematically characterize the interaction network in vivo using two distinct models of polarity: asymmetric division of the one-cell embryo, and stem-cell-like divisions of a multicellular epithelium (in collaboration with M. Wildwater and S. van den Heuvel). M. Boxem, Z. Maliga, N. Klitgord, N. Li, I. Lemmens, M. Mana, L. de Lichtervelde, J. D. Mul, D. van de Peut, M. Devos, N. Simonis, M. A. Yildirim, M. Cokol, H. L. Kao, A. S. de Smet, H. Wang, A. L. Schlaitz, T. Hao, S. Milstein, C. Fan, M. Tipsword, K. Drew, M. Galli, K. Rhrissorrakrai, D. Drechsel, D. Koller, F. P. Roth, L. M. Iakoucheva, A. K. Dunker, R. Bonneau, K. C. Gunsalus, D. E. Hill, F. Piano, J. Tavernier, S. van den Heuvel, A. A. Hyman, and M. Vidal, A protein domain-based interactome network for C. elegans early embryogenesis. Cell, 2008. 134(3): p. 534-545. .

Mecenas, D. et al. (2013) International Worm Meeting "Alternative 3'UTRs: A Mechanism for Post-transcriptional Regulation in C. elegans Germline & Early Embryo."

Reboul, J., Gutwein, M., Ahmed, R., Polanowska, J., Gunsalus, K., Mecenas, D., Piano, F.

[International Worm Meeting, 2013]

Proper spatio-temporal control of gene activity is vital for animal development. In the C. elegans germline and early embryo, this occurs mainly by post-transcriptional regulation (PTR) via the three-prime untranslated region (3'UTR). PTR depends on cis-regulatory sequences and trans-acting factors, including RNA binding proteins (RBPs) and small regulatory RNAs, which influence the stability, translation, and localization of mRNA. Experimental analysis of PTR is technically challenging, and in vivo studies of the molecular mechanisms by which 3' UTRs regulate gene activity are limited. Sequence analysis of the 3'UTR landscape in C. elegans indicates that about half of the protein-coding genes are alternatively polyadenylated (Mangone et al., Science 2010; Jan et al., Nature 2011). Therefore, alternative polyadenylation (APA) can result in mRNA transcripts with 3'UTRs of varying lengths and regulatory potential. In the germline, 3'UTR-mediated PTR is the primary mechanism of gene regulation (Merritt et al., Curr Biol 2008). In addition, a number of key RBPs whose activities are crucial for development are expressed in spatio-temporally restricted regions. Our goal is to address the question of correlation between the availability of regulatory elements within the 3'UTR and their cognate trans-acting factors. By combining data on 3'UTR isoforms with iPAR-CLIP of GLD-1 (Jungkamp et al., Mol Cell 2011), we found 38 genes that experience APA and contain binding sites for GLD-1 in the variable 3'UTR region. To test the idea that individual transcripts may escape PTR by selective expression and/or degradation of alternative 3'UTRs in the germline and early embryo, we developed assays to detect and quantify them during development. We have cloned a subset of these 3'UTRs into a two-color in vivo reporter system that will provide a readout of 3'UTR-mediated PTR. Combining these expression assays with mutational analysis of 3'UTRs will allow us to assay the relative contributions of alternative isoforms and regulatory elements to specific spatio-temporal expression patterns during development.

Maelle Jospin et al. (2001) International C. elegans Meeting "Basic electrophysiological properties of body wall muscle from the Nematode C elegans."

Maelle Jospin, Laurent Segalat, Bruno Allard

[International C. elegans Meeting, 2001]

Electrophysiological properties of striated muscle cells were investigated with the patch clamp technique in the Nematode C elegans . Worms were immobilised with cyanoacrylate glue and longitudinally incised using a tungsten rod sharpened by electrolysis. Recording pipettes were sealed on GFP-expressing body wall muscle cells. In the whole cell configuration, under current clamp conditions, in the presence of Ascaris medium in the bath and K-rich solution in the pipette, no action potential could be induced in response to current injection. Under voltage clamp control and in the same ionic conditions, depolarizations above -30 mV from a holding potential of -70 mV gave rise to outward K currents. Outward K currents resulted from two components, one fast inactivating component blocked by 4-aminopyridine, one delayed sustained component blocked by tetraethylammonium. In the presence of both blockers, an inward Ca current was revealed and inhibited by cadmium. Single channel recording using the inside-out configuration revealed the existence of a Ca-activated Cl channel and a Ca-activated K channel. Single channel experiments are currently performed to characterise voltage-gated conductances at the unitary level.

Fitch, D. et al. (2013) International Worm Meeting "RhabditinaDB: online database for wild worms."

Kiontke, K., Fitch, D.

[International Worm Meeting, 2013]

New species of rhabditid nematodes are being discovered at an increasing rate. In Caenorhabditis alone, more species have been discovered in the last decade than in the previous 100 years since C. elegans was described. Although this discovery provides more candidate species for comparative biology and genomics, it poses a challenge for taxonomists and for keeping track of comparative data. In response to this challenge, we have created RhabditinaDB, a curated, online, open-access database to provide information on all known rhabditid nematodes, including data on taxonomy, phylogeny, distribution, living and non-living resources such as strains and type specimens, images (including DIC stacks, electron micrographs, and camera lucida drawings), species descriptions and relevant literature, phenotypic character data and molecular sequences. Additionally, we have implemented tools for querying the data, such as keyword searching, BLAST, PhenoBLAST (modified from Gunsalus et al., 2004) and a species-comparison page. Currently, the database shows information for all known species in genus Caenorhabditis, but will be expanded in the coming years to show data for all rhabditid species. Some problems in rhabditid systematics have arisen from the difficulty of obtaining taxonomic materials, lack of good keys or ways to compare new species to those already described. Specifically, there has been a high rate of synonymous descriptions and the accumulation of newly discovered but undocumented species. Our database should mitigate these problems by providing as much data as possible via one resource, along with a recent taxonomy (Sudhaus 2011), based primarily on our molecular phylogeny (Kiontke et al., 2007, 2012). Not only will RhabditinaDB facilitate taxonomy, but it will also provide data on rhabditid biodiversity. This in turn should facilitate phylogenetically informed taxon choices for comparative studies and genomics. As RhabditinaDB is a work in progress, we welcome suggestions for improvements, and data submissions. RhabditinaDB can be accessed at: http://wormtails.bio.nyu.edu/Databases . Thanks to M. Katari, J. Lorenzana, NYU Bobst Library, W. Sudhaus. References: Gunsalus et al. 2004, Nucl. Acids Res. 32:D406-D410; Sudhaus 2011, J. Nematode Morphol. Syst. 14(2):113-178; Kiontke et al. 2007, Curr. Biology 17(22):1925-1937.

Chung, G. et al. (2019) International Worm Meeting "The function of meiosis I genes in the parthenogenic nematode Diploscapter pachys."

Piano, F., Gunsalus, K., Zegar, C., Fitch, D., Chung, G., Baugh, R., Kiontke, K., Fradin, H.

[International Worm Meeting, 2019]

Asexual reproduction has arisen independently multiple times in the phylum Nematoda and requires numerous modifications to the reproductive program to produce a viable diploid zygote without fertilization of the oocyte. One possible modification may involve tampering with meiotic recombination, whose loss ensures the continued transmission of heterozygosity in the absence of genetic material from a second gamete. To test this hypothesis, we sequenced the genome of the parthenogenic nematode Diploscapter pachys (1), whose oocytes undergo a single meiosis II-like division and continue on to mitotic divisions without prior chromosome synapsis and apparent recombination. Strikingly, the D. pachys genome reveals extensive rearrangement among neighboring ancestral regions with high heterozygosity across most regions. Additionally, cytological observations indicate the diploid genome is contained within a single pair of chromosomes. However, in the D. pachys genome and transcriptome, we found several full-length coding sequences normally required for meiotic recombination in meiosis I: Dpa-spo-11, the ortholog of the spo-11 gene required for the generation of meiotic double-strand breaks (DSBs), and Dpa-dmc-1, the ortholog of the dmc-1 gene required for the repair of DSBs by homologous recombination in meiosis I. In order to reconcile these findings, we are developing tools to detect meiotic DSBs and their possible repair pathways in D. pachys. This will clarify the role of meiotic recombination in the maintenance of heterozygosity. Furthermore, we are improving the genome assembly to better understand the ancient chromosomal fusion events which led to the unusual unichromosomal genome architecture of D. pachys. The temporal sequence and causal relationships of molecular events leading to altered meiosis, egg autoactivation, and chromosomal fusion are yet to be determined. Elucidating the nature of these changes would provide a deeper understanding of the ways in which animals can modify their reproductive programs during evolution from gonochorism to parthenogensis.

K Omata et al. (1999) International C. elegans Meeting "Habituation of C. elegans for the touch sensitivity"

K Kawamura, F Yonezawa, K Omata

[International C. elegans Meeting, 1999]

In order to study the habituation of C. elegans for the touch sensitivity, we carry out computer simulations, in which the neural circuit is formed by making use of the data table constructed recently by Oshio et al [1]. The i -th neuron is connected with the neighboring j -th neuron through the coupling strength K ij , which is varied dynamically by the Hebb rule. Note that K ij is not necessarily equal to K ji because there are one-way connections between the neurons by chemical synapses. As a reference state, we first deal with the neural circuit consisting only of the neurons ALM, AVM, PLM, PVM, AVA, AVB, PVC, AVD, A and B, that are related to the forward and backward movement directly. We give periodic stimuli to the sensory neurons PLM, PVM, and monitor the response of the motor neuron A. We find that the frequency of the response decreases with time, which indicates that the habituation to the touch sensitivity actually takes place. As one deviation from the reference state, we kill the inter-neuron AVD, and perform the same analysis described in the above. There is a tendency that the decay of the response curve becomes faster, and the habituation is enhanced. As the other deviations, there are several possibilities of killing the inter-neurons AVA, AVB, PVC and/or AVD. We discuss the enhancement of the habituation in relation with the recent experimental results by Hosono. [1.] K. Oshio, S. Morita, Y. Osana and K. Oka; C. elegans synaptic connectivity data'', Technical Report, CCEP, Keio Future No.1 (1998)