Khavari AR et al. (1997) International C. elegans Meeting "Dominant mutations affecting dauer development."

Baird SE, Grunwald WC, Khavari AR

[International C. elegans Meeting, 1997]

Dauer developement is, in part, regulated by a TGF-beta like signalling pathway. The ligand, type II receptor, and type I receptor for this pathway are coded for by the daf-7, daf-4, and daf-1 genes, respectively (Pen et al. Science 274, 1389-1391, 1996; Estevez et al., Nature 365, 644-649, 1993; Georgl et al., Cell 61, 635-645, 1990). Loss-of-function mutations in these genes result in a temperature-sensitive dauer constitutive (Daf-C) phenotype. To identify additiona1 components of this pathway we have conducted screens for dominant mutations that suppress the Daf-C phenotype of daf-7. Over 1.5 million F1 worms have been screened and eleven dominant suppressors have been obtained. One of these suppressor, bd17, has been has been mapped to the left end of LG III and may be an intragenic revertant of daf-7. Another, bd27, has been mapped to the center of LG X. The map positions of the remaining nine suppressors have not yet been determined. We currently are conducting additional screens and characterizng the mutations alreaty obtained.

Henry Schaefer et al. (2005) International Worm Meeting "KEL-8, a novel Kelch-like protein, is required for glutamate receptor degradation."

Jaegal Shim, Christopher Rongo, Henry Schaefer

[International Worm Meeting, 2005]

In the central nervous system, glutamate receptor insertion into and removal from the postsynaptic membrane is an important mechanism for changes in synaptic strength. Glutamate receptor localization is a stepwise process that includes transport from the cell body to postsynaptic specializations in the neurite, exocytosis into the membrane, endocytosis, and eventual degradation. Using a visually-based, forward genetic screen with animals expressing a fluorescently-tagged GLR-1 receptor subunits, we isolated mutants with mislocalized glutamate receptors. One of the genes found to be required for receptor turnover produces a Kelch-like protein we are calling KEL-8. In kel-8 mutants, unlocalized receptor fills the ventral nerve cord. Other synaptic proteins are properly localized in kel-8 mutants, suggesting that KEL-8 is not required for general cell polarity or synapse formation per se. We find that KEL-8 is expressed in neurons and is localized to punctate structures in the neurite, adjacent to GLR-1 postsynaptic clusters. A possible actin-binding protein, KEL-8 has previously been found to bind proteasome subunits (1). We find that KEL-8 colocalizes with the proteasome in the ventral cord neurites. It has been shown that ubiquitination of glutamate receptors causes their degradation, and overexpression of monoubiquitin reduces the abundance of glutamate receptor in the neurite (2). However, the accumulation of receptor in kel-8 mutants is resistant to this ubiquitin-mediated turnover. This evidence suggests a novel function for a Kelch-like protein in the ubiquitin-mediated degradation of glutamate receptors by the proteasome. The general role of ubiquitin in GLR-1 degradation will also be discussed. 1.A. Davy et al., EMBO Rep 2, 821-8 (Sep, 2001). 2.M. Burbea, L. Dreier, J. S. Dittman, M. E. Grunwald, J. M. Kaplan, Neuron 35, 107-20 (Jul 3, 2002).

Volkers, R. J. M. et al. (2013) International Worm Meeting "Gene-environment interactions drive genomic and transcriptomic diversity in wild Caenorhabditis elegans populations."

Chen, W., Schulenburg, H., van Hellenberg hubar, C. J., Coopman, R., Sterken, M. G., Kammenga, J. E., Volkers, R. J. M., Braekman, B. P., Snoek, L. B.

[International Worm Meeting, 2013]

RJMV and LBS contributed equally to this work. Background: Analyzing and understanding the relationship between genotypes and phenotypes is at the heart of genetics. Research in the nematode C. elegans has been instrumental for unravelling many genotype-phenotype relations. But almost all studies, including forward and reverse genetic screens, are dominated by investigations in one canonical single strain. In order to explore the full potential of the natural genetic variation and evolutionary context of the genotype-phenotype map, it is important to study these relations in wild populations. Results: We used multiple wild strains freshly isolated from local habitats to investigate the gene sequence polymorphisms and a multitude of phenotypes including the transcriptome, fitness and behavioural traits. These showed a direct link with the original habitat of the strains. The separation between the isolation sites was prevalent on all chromosomes, chr. V contributing the most. These results were supported by a differential food preference of the wild isolates for naturally co-existing bacterial species. Moreover, we show that genomic and transcriptomic diversity was driven by genes involved in gene-environment interactions, like c-type lectins and fbox genes. Conclusions: Importantly, where wild C. elegans strains show a broad range of genotype-phenotype relations the widely studied canonical genotype N2 covers only a diminutive part of the myriad of genotype-phenotype relations which are present in the wild. Funding: RJMV and LBS: NWO-ALW (project 855.01.151) NEMADAPT, RC and BPB: ESF-EEFG (09-EuroEEFG-FP-002 / G.0998.10N) NEMADAPT. JEK: ERASysbio-plus ZonMW project GRAPPLE (project nr. 90201066). MGS: Graduate School Production Ecology & Resource Conservation. WC and HS: NEMADAPT (DFG grant SCHU 1415/11-1).

Hodul, Molly et al. (2017) International Worm Meeting "Regulation of the deubiquitinating enzyme USP-46 and the glutamate receptor GLR-1 by WD40 repeat proteins."

Juo, Peter, Hodul, Molly, Dahlberg, Caroline L.

[International Worm Meeting, 2017]

Ubiquitin-mediated endocytosis and degradation of glutamate receptors controls their synaptic abundance and is implicated in modulating synaptic strength, learning, and memory. Ubiquitination of GLR-1, a C. elegans AMPA-type glutamate receptor, promotes receptor internalization and degradation in the lysosome (1). Ubiquitination can be reversed by deubiquitinating enzymes (DUBs). We previously found that the DUB Ubiquitin-Specific Protease 46 (USP-46) regulates GLR-1 levels (2). USP-46 removes ubiquitin from GLR-1 and likely acts at endosomes to protect GLR-1 from lysosomal degradation. Consistently, usp-46 null mutants have decreased GLR-1 at synapses in the ventral nerve cord (VNC) and corresponding defects in GLR-1-dependent behaviors (2). In contrast, overexpression of usp-46 does not have the converse effect, suggesting that USP-46 activity is tightly controlled in neurons. Very little is known about how DUBs are regulated in vivo. Two WD-40 repeat proteins, WDR-48 and WDR-20, were previously shown to interact with USP-46 in mammalian cells (3-5). We showed that the C. elegans homologs of these WDR proteins bind USP-46 and stimulate DUB catalytic activity in vitro (6). Overexpression of wdr-48 and wdr-20 in neurons in vivo results in increased synaptic levels of GLR-1, and this effect is further enhanced by co-expression of usp-46 (6). Here we investigate several mechanisms by which the WDR proteins regulate USP-46 and GLR-1. First, we found that overexpression of wdr-48 and wdr-20 increase the abundance of USP-46 in HEK cells and in neurons in vivo. We will take advantage of point mutations known to disrupt the binding between each WDR and USP-46 to test if their direct interaction is required for the increased levels of USP-46. Second, we have preliminary evidence suggesting that WDR-48 recruits USP-46 to endosomes where it is presumed to act on ubiquitinated GLR-1. Finally, we recently found that wdr-20 but not wdr-48 transcription is regulated by chronic synaptic activity-blockade, which has previously been shown to increase synaptic GLR-1 (7). Thus, WDR-48 and WDR-20 are emerging as key regulators that appear to use multiple mechanisms to control USP-46 and its effects on GLR-1. 1. Burbea et al. (2002) Neuron 2. Kowalski et al. (2011) J Neurosci 3. Sowa et al. (2009) Cell 4. Cohn et al. (2009) JBC 5. Kee et al. (2010) JBC 6. Dahlberg & Juo (2014) JBC 7. Grunwald et al. (2004) PNAS

Lars Dreier et al. (2003) International Worm Meeting "LIN-23, a subunit of an SCF-type ubiquitin ligase, regulates the abundance of synaptic glutamate receptors"

Michelle Burbea, Josh Kaplan, Lars Dreier

[International Worm Meeting, 2003]

An important feature of the signaling between neurons is the activity-dependent change in synaptic strength. One mechanism to increase or decrease synaptic strength is to increase or decrease the number of neurotransmitter receptors at the postsynaptic membrane. We have previously shown that ubiquitination of the glutamate receptor GLR-1 regulates the abundance of synaptic receptors and thereby the strength of glutamatergic synapses (1). Consistent with these results, we isolated two alleles of lin-23 in a screen for mutations that change the abundance of GLR-1 in the ventral cord. The lin-23 gene encodes an F-box protein, which is the substrate binding subunit of an SCF-type ubiquitin ligase (2). lin-23 mutants have aberrant cell cycle regulation that results in extra larval cell divisions (2). We found that lin-23 mutations cause a dramatic increase of GLR-1 in the ventral cord. The GLR-1 localization defect of lin-23 mutants can be rescued by expressing LIN-23 in the GLR-1-expressing interneurons whereas expression of a dominant-negative form of LIN-23 in these cells mimics the lin-23 mutant phenotype. Thus, LIN-23 acts cell autonomously in these interneurons to control the abundance of GLR-1 in the ventral cord. We previously reported that over-expression of ubiquitin in the ventral cord interneurons decreases the abundance of GLR-1 at synapses and decreases the number of GLR-1-containing synapses. Mutations in lin-23 partially suppress the effects of ubiquitin over-expression. These results suggest that a ubiquitin ligase containing the LIN-23 F-box protein regulates GLR-1 containing synapses. We used two approaches to test whether LIN-23 is involved in the ubiquitination of GLR-1. First, we measured ubiquitination of GLR-1 in wild-type animals and in animals expressing a dominant-negative LIN-23. Expression of a dominant-negative LIN-23 did not affect ubiquitination of GLR-1. Second, we used a non-ubiquitinated GLR-1 (4KR-GLR-1) that is more abundant at synapses than wild-type receptor. Expression of 4KR-GLR-1 in lin-23 mutants resulted in an additive phenotype for receptor abundance in the ventral cord. Taken together, these results suggest that GLR-1 is not a target of the LIN-23-containing ubiquitin ligase. We are currently attempting to identify the target of LIN-23 that is involved in regulating the abundance of GLR-1.1. Burbea M, Dreier L, Dittman JS, Grunwald ME, Kaplan JM.2002.Neuron.35:107; 2. Kipreos ET, Gohel SP, Hedgecock EM. Development.2000;127:5071

Nicholas Duclos et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Identifying Genes Required for Signaling via the CAM-1 Receptor Tyrosine Kinase"

Nicholas Duclos, Andres Maricq, Michael Francis, Penelope Brockie

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Signaling at synapses requires that cells within the nervous system adopt the correct cell identity. Thus, both pre- and post-synaptic cells must express a specific compliment of molecules and establish the appropriate synaptic connections. We would like to understand how the C. elegans Ror receptor tyrosine kinase (RTK), CAM-1, contributes to these processes. Ror RTKs are highly expressed in the vertebrate nervous system as well as other tissues, yet the mechanism of signaling by these RTKs remains unclear. cam-1 encodes the only Ror RTK in C. elegans and is normally expressed in both the nervous system and in body wall muscles. In neurons, it has been associated with EGL-20, a Wnt signaling molecule, in regulating nervous system development (Forrester et al.). At the neuromuscular junction, it has been implicated in the stabilization and localization of the acetylcholine receptor subunit ACR-16 (Francis et al.). Overexpression of CAM-1 in either muscles or neurons produces dramatic overgrowth of cellular processes. In order to identify gene products that regulate Ror RTKs, we have initiated a forward screen for modifiers of CAM-1 function that takes advantage of this overexpression phenotype. Overexpression of CAM-1 in the command interneurons produces a dramatic change in cell morphology that is easily identifiable by the presence of numerous highly branched ectopic neuronal outgrowths. To identify genes that function in the same pathway as cam-1, we are screening for suppressors of this phenotype. We expect to find genes that encode upstream regulators of CAM-1 signaling, such as potential ligands, as well as downstream components of the CAM-1 signaling pathway. The identification of these genes will yield valuable insights in to the mechanisms of Ror RTK signaling. Francis MM, Evans SP, Jensen M, Madsen DM, Mancuso J, Norman KR, Maricq AV. The Ror Receptor Tyrosine Kinase CAM-1 Is Required for ACR-16-Mediated Synaptic Transmission at the C. elegans Neuromuscular Junction. Neuron 46: 581-594Forrester WC, Kim C, Garriga G. The C. elegans Ror RTK CAM-1 collaborates with EGL-20/Wnt to regulate cell migration. 168: 1951-1962

Wang, Lin et al. (2017) International Worm Meeting "Dissecting the roles of the ADAM10 metalloprotease SUP-17 in the BMP signaling pathway in Caenorhabditis elegans."

Liu, Jun, Liu, Zhiyu, Shi, Herong, Wang, Lin

[International Worm Meeting, 2017]

BMPs (Bone Morphogenetic Proteins) belong to the TGFbeta superfamily of ligands, and mediate a highly conserved signal transduction cascade. Upon ligand binding, type II receptors phosphorylate type I receptors, which in turns phosphorylate R-Smads (receptor regulated Smads). Phosphorylated R-Smads then complex with co-Smad (common mediator smad) and enter the nucleus to regulate gene transcription with different co-factors. BMPs play important roles in developmental and physiological processes. Malfunction of the pathway in humans can cause various disorders, such as skeleton diseases, heart diseases and cancers. So it is critical to strictly regulate the level of BMP signaling spatiotemporally. Using a highly specific genetic screen, we have identified several modulators of the BMP pathway in C. elegans. These include the Neogenin homolog UNC-40 [1], two paralogous tetraspanin proteins, TSP-12 and TSP-14, and an ADAM10 metalloproteinase (A Disintegrin and Metalloproteinase 10) SUP-17 [2]. We use the CRISPR/Cas9 system and tagged the endogenous TSP-12, SUP-17 and UNC-40 proteins with different fluorescence tags. We found that TSP-12 is localized to the cell surface and intracellular vesicles, and that TSP-12 can bind to SUP-17 and promote its cell surface localization. We have genetic evidence and preliminary biochemical evidence showing that UNC-40 is one, but not the only, substrate of SUP-17 in BMP signaling. We are currently identifying additional substrate(s) of SUP-17 in BMP signaling. Our work highlights the importance of intracellular signaling and protease processing in the regulation of BMP signaling. [1] Tian C, Shi H, Xiong S, Hu F, Xiong WC, Liu J. The neogenin/DCC homolog UNC-40 promotes BMP signaling via the RGM protein DRAG-1 in C. elegans. Development. 2013;140(19):4070-80. doi: 10.1242/dev.099838. PubMed PMID: 24004951; PubMed Central PMCID: PMCPMC3775419. [2] Wang L, Liu Z, Shi H, Liu J. Two Paralogous Tetraspanins TSP-12 and TSP-14 Function with the ADAM10 Metalloprotease SUP-17 to Promote BMP Signaling in Caenorhabditis elegans. PLoS Genet. 2017;13(1):e1006568. doi: 10.1371/journal.pgen.1006568. PubMed PMID: 28068334; PubMed Central PMCID: PMCPMC5261805.