Beverly, Matthew et al. (2011) International Worm Meeting "Degeneracy and neuromodulation in the thermosensory circuit regulates robustness in thermosensory behaviors in C. elegans."

Bell, Harold, Anbil, Sriram, Beverly, Matthew, Sengupta, Piali

[International Worm Meeting, 2011]

Animals must exhibit physiologically important behaviors under a wide range of conditions. The mechanisms by which neural circuits generate robust behaviors are not well understood. C. elegans maintains optimal body temperature by regulating locomotory behavior. When exposed to temperatures greater than their cultivation temperature (Tc), C. elegans moves towards colder temperatures in a behavior called negative thermotaxis. The AFD and AWC sensory neurons are thermosensory and are required for negative thermotaxis under the limited set of conditions that have been examined to date. However, whether these neurons contribute similarly to negative thermotaxis under all conditions, or whether additional circuit components are recruited under specific conditions, are unknown. By analyzing the requirement of different sensory neurons in mediating negative thermotaxis under conditions of varying Tc and gradient temperature ranges, we have identified the ASI chemosensory neurons as a third thermosensory neuron type. Different combinations of the AFD, AWC and ASI thermosensory neuron types are necessary and sufficient for negative thermotaxis under different conditions. We find that the ASI neurons respond to changes in temperature within an operating range whose bounds are defined by Tc. Interestingly, this operating range of ASI is altered in mutants affecting neuropeptidergic signaling suggesting that neuromodulation, possibly from AFD, coordinates thermosensory neuron response ranges to ensure a coherent behavioral output. In preliminary work, we have identified neuropeptide receptor and ligand genes, mutations in which alter the temperature response range of ASI, and are currently characterizing their behavioral phenotypes and sites of action. Our work suggests that multiple circuit configurations can act degenerately to generate the same behavior under different conditions, and emphasize the importance of defining context when describing neuronal contributions to a behavior.

Li, Weixun et al. (2015) International Worm Meeting "Regulator of Calcineurin (RCAN-1) in AFD neurons regulates thermotaxis behavior."

Lee, Sun-Kyung, Bell, Harold, Ahnn, Joohong, Li, Weixun

[International Worm Meeting, 2015]

Regulator of calcineurin (RCAN) is a calcineurin-interacting protein that inhibits calcineurin phosphatase. In this study, we found that rcan-1 deletion mutants exhibited cryophilic behavior dependent on tax-6, which was rescued by expressing rcan-1 in AFD neurons. Interaction between RCAN-1 and TAX-6 requires the conserved PxIxIT motif of RCAN-1, without which thermotactic behavior could not be fully rescued. Calcium imaging studies indicate that AFD neurons in rcan-1 and tax-6 mutants are abnormal. In addition, the loss of crh-1/CREB suppressed the thermotaxis phenotypes of rcan-1 and tax-6 mutants, suggesting that crh-1 may play a role downstream of the rcan-1/tax-6 pathway. Taken together, our results suggest that rcan-1 is an inhibitory regulator of tax-6 in AFD neurons, and that it acts in the formation of thermosensory behavioral memory in C. elegans. .

Takeishi*, Asuka et al. (2015) International Worm Meeting "AFD-specific receptor guanylyl cyclases can confer temperature responses onto diverse cell types."

Takeishi*, Asuka, Sengupta, Piali, Bell, Harold W., Hapiak, Vera M., Yu*, Yanxun V.

[International Worm Meeting, 2015]

C. elegans exhibits complex experience-dependent thermosensory behaviors on spatial thermal gradients. AFD has been identified as the major thermosensory neuron type, and AFD-ablated worms exhibit strong defects in thermotaxis behaviors. Measurements of temperature-evoked intracellular calcium dynamics and thermoreceptor current have shown that AFD responds to thermal variation at temperatures above a threshold temperature (T*) that is set by the animal's cultivation temperature (Tc). Thermotransduction in AFD is mediated by cGMP signaling, and requires the functions of the receptor guanylyl cyclases (rGCs), GCY-8, GCY-18 and GCY-23, the cGMP-dependent phosphodiesterase PDE-2, and the TAX-2 and TAX-4 cGMP-gated channels. However, the molecular mechanisms by which AFD senses temperature are still unknown.Previous studies have shown that many rGCs function as sensors for specific stimuli, suggesting that the AFD-expressed rGCs may themselves act as thermosensors. We found that misexpression of GCY-8, GCY-18 and GCY-23 in the non-thermosensory AWB and ASE chemosensory neurons is sufficient to confer warming-dependent changes in intracellular calcium (also see abstract by Yu, Takeishi et al). We further expressed each rGC singly or in combination in these neurons, and found that expression of GCY-23 alone confers temperature responses onto AWB and ASE in the physiological temperature range, whereas expression of GCY-18 confers responses to higher temperatures. Interestingly, in contrast to AFD, the T* of AWB and ASE misexpressing AFD-specific rGCs is not modulated by Tc, suggesting that the observed Tc -dependent plasticity in AFD thermosensory response threshold may be mediated by AFD-specific mechanisms. To ask whether misexpression in cell types lacking endogenous cGMP signaling pathways would also confer thermosensory responses, we next misexpressed the rGCs in vulval muscles. We found that vulval muscles misexpressing GCY-23 as well as TAX-2/TAX-4 also exhibit responses to temperature variation. Our results suggest that AFD-specific rGCs can confer thermosensory responses onto multiple neuronal and non-neuronal cell types, and raise the possibility that these proteins may be bona fide thermosensors in C. elegans.* - equal contributions.

Katherine M Walstrom et al. (2001) International C. elegans Meeting "Biochemical Characterization of C. elegans Malate Dehydrogenase"

Danny Gonzalez, Katherine M Walstrom

[International C. elegans Meeting, 2001]

In my laboratory, we are overexpressing and biochemically characterizing C. elegans malate dehydrogenase (MDH), the last enzyme in the citric acid cycle. This enzyme, encoded by putative gene F20H11.3, appears to be the mitochondrial form of MDH, since it contains a mitochondrial import presequence. We have overexpressed and purified a version of this enzyme with the mitochondrial import presequence removed at the predicted cleavage site of the mitochondrial processing protease. The purified MDH enzyme has malate dehydrogenase activity that follows Michaelis-Menten kinetics when plotted versus oxaloacetate concentration, and the resulting Km is similar to the Km values for other MDH enzymes. Interestingly, the temperature dependence of the enzyme is a bell-shaped curve with a peak at approximately 30degC, suggesting that the enzyme is adapted to the growing temperature of C. elegans . We are grateful to Yuji Kohara for providing the cDNA clone of MDH.

David R. Bell et al. (2006) European Worm Meeting "The Function of Latrophilin in C. elegans"

S. van der Poel, P.N.R. Usherwood, D. Kendall, J. Davy, A. Ademola, I.R. Duce, D. de Pomerai, David R. Bell, I. R. Mellor

[European Worm Meeting, 2006]

David R. Bell, A. Ademola, J. Davy, S. van der Poel, D. Kendall, P.N.R. Usherwood, I.R. Duce, I. R. Mellor and D. de Pomerai. Black Widow spider venom (BWSV) contains latrotoxins that induce catastrophic neurotransmitter release in mammals, and is known to bind with high affinity to three neural proteins in mammals, including latrophilin. We have investigated C. elegans as a model system for studying the function of these proteins, and their role in regulating neurotransmitter release by latrotoxins.. We have shown that latrophilin is required for the lethality of BWSV in C. elegans by RNAi experiments. We now present data on the characterisation of null mutants of the C. elegans latrophilin, lat-1, illustrating the function of this gene in C. elegans, and show that lat-1 nulls are resistant to emodepside. The expression of latrophilin-GFP constructs is examined, and compared with the phenotypes seen in null mutants. Genetic interactions between endogenous signaling pathways and the lat-1 nulls have been examined by the construction of double mutant worms, revealing novel facets of lat-1 function.

Sarah E McCarthy et al. (2003) International Worm Meeting "Searching for proteins that interact with FER-1"

Samuel Ward, Sarah E McCarthy, Nicole L Washington

[International Worm Meeting, 2003]

The fusion of membranous organelles to the plasma membrane of an activating spermatid requires FER-1, a sperm-specific protein. By sequence homology, fer-1 is related to the human Limb Girdle Muscular Dystrophy gene, Dysferlin. FER-1 is a 237 kD protein comprised of five C2 domains, a dysferlin domain, and a transmembrane domain very near the C-terminus. In general, C2 domains may possess any or all of the following: calcium binding, phospholipid binding, protein-protein interactions and hetero- and homo-oligomerization. C2 domains are typically 100 amino acids and form a beta-sandwich structure. The DysfN/C domain is largely uncharacterized as searching for the DysfN/C profile results in a few predicted C. elegans and human proteins, and a yeast peroxisomal protein. To test the hypothesis that FER-1 interacts with sperm proteins through its C2 and Dysf domains, we are utilizing a LexA-based yeast two-hybrid system. The domains have been amplified by PCR and cloned into pEG202 to be used as bait in the screen. The sperm-specific cDNA library was a generous gift from Harold Smith. Although there are numerous possibilities for proteins that may be found to interact with the FER-1 domains, there are a few proteins that we hypothesize will be identified in this screen. Given that FER-1is suspected to be involved in membrane fusion events, possible proteins may include SNAREs or other members of the membrane fusion machinery. For example, synaptotagmin contains two C2 domains, the first of which promotes binding with syntaxin. In addition, preliminary immunolocalization of FER-1 and CAV-1 in spermatids combined with the presence of a caveolin-binding sequence in one of the C2 domains make caveolin a strong candidate for interaction.

Craig W LaMunyon (2003) International Worm Meeting "Mutations that disrupt uniparental mitochondrial inheritance: a forward and reverse approach"

Craig W LaMunyon

[International Worm Meeting, 2003]

Paternal mitochondria are universally destroyed in developing embryos. Recent evidence indicates that mammalian sperm mitochondria are marked with ubiquitin, which directs their degradation in the fertilized egg. My laboratory is currently investigating paternal mitochondrial degradation in C. elegans, testing the hypothesis that sperm mitochondria are tagged for degradation. In a forward genetic analysis, we have screened nearly 2,000 F2 males for EMS induced mutations that allow sperm mitochondrial survival. Paternal mitochondrial DNA is marked with a 3,000 bp deletion (strain UaDf-5, kindly provided by Dr. Bernard Lemire at the University of Alberta, Edmonton, Canada). Paternal mitochondria are detected using a nested PCR assay, and we have generated 4 potential mutants, although homozygous strains have not yet been obtained. These preliminary results suggest that as in mammalian sperm, C. elegans sperm mark their mitochondria for degradation. Identification of the mutated genes will shed light on the mechanism of degradation. We are also taking a reverse genetic approach to examine the role of ubiquitin-associated genes that are expressed solely in sperm. Recent microarray analyses have identified 4 such genes. In collaboration with Dr. Harold Smith (Center for Advanced Research in Biotechnology, Rockville, MD), we undertaking targeted gene knockouts of these 4 genes. We have obtained a deletion mutation in C34F11.1, a putative E3 ubiquitin ligase based on a conserved RING finger domain. Worms with this mutation are fertile but show reduced fecundity, and we will soon be testing them for paternal mitochondrial survival. Should mutations in any of these 4 ubiquitin-associated sperm-specific genes block the degradation of paternal mitochondria, a ubiquitin mechanism will be implicated. Finally, examination of the offspring that inherit paternal mitochondria will allow us to test hypotheses for the evolution of maternal-only inheritance.

Girgenrath, J.K. et al. (2019) International Worm Meeting "Investigating the interaction between germline specific DEAD-box helicase VBH-1 and CSR-1."

Ghanta, K.S., Mello, C.C., Girgenrath, J.K.

[International Worm Meeting, 2019]

The Argonaute CSR-1 localizes to the perinuclear germline nuage, P-granules where it is required for RNA-induced epigenetic activation (RNAa), a process thought to protect endogenous germline transcripts from RNA-induced epigenetic silencing (RNAe) (Seth et al., 2013). To better understand CSR-1 function we identified interacting proteins through immunoprecipitation and Mass-Spectrometry analysis. This analysis revealed many peptides corresponding to the Vasa-Bell homolog, VBH-1, a DEAD-box protein previously shown to localize in P-granules (Salinas et al., 2007). The C. elegans germline expresses a wealth of DEAD-box proteins with widely varying functions. These include the GLH-1,2,3 and 4 proteins that, like VBH-1, co-localize in P-granules but instead of CSR-1, preferentially engage distinct Argonaute proteins. Although the exact function of VBH-1 has not been determined, our preliminary data shows that vbh-1 null mutants are viable but have significantly reduced brood sizes at 20? and 25? suggesting that it promotes temperature-dependent fertility. Interestingly, point mutants with lesions in the DEAD-box (DQAD mutants) exhibit gain-of-function or possibly antimorphic phenotypes that compromise fertility even more severely than do complete null mutants. Neither the null nor the DQAD mutants exhibit defects in CSR-1 localization. Also, vbh-1 null mutants did not have compromised RNAa activity, suggesting it is not required for this function of CSR-1. Currently, experiments are being conducted to characterize the protein-expression levels of CSR-1 targets in vbh-1 mutants. In the future, VBH-1 cross-linking IP (CLIP) experiments will be carried out to characterize the mRNA-binding profile of VBH-1. In addition, we are profiling CSR-1-associated small RNAs in wild-type and VBH-1 mutants to determine whether VBH-1 promotes the robustness of mRNA targeting at elevated temperatures. Using data generated through these experiments, we hope to obtain a greater understanding of CSR-1 and VBH-1 function in promoting fertility.

Shan Ge et al. (2001) International C. elegans Meeting "Male Mating Behavior and the Role of UNC-55, A Nuclear Receptor"

Shan Ge, Dorothy Sojka, Bill Walthall

[International C. elegans Meeting, 2001]

unc-55 encodes a transcription factor that is a member of the nuclear receptor gene family. unc-55 mutant males are unable to mate successfully. The relatively mild defect in coordination is unlikely to be responsible for the defect in male-mating behavior. Using an unc-55::gfp transgenic strain, gfp expression was observed in one neuron, a muscle and the four spicule sheath cells in the male tail. The spicule sheath cells, which normally have a dumb-bell shape, are stretched when the spicules are extended. The pattern of expression in unc-55 mutant males was indistinguishable from wild-type males. Observation of unc-55 mutant male behavior revealed that males exhibit normal chemotaxis in locating hermaphrodites, move backward along the body of hermaphrodites and make appropriate turns but mutants do not copulate. Further observation of unc-55 males revealed that often the paired spicules are extended in the absence of hermaphrodites, this is rarely observed in wild-type males. One approach that we are using to investigate the relationship between the cells expressing unc-55::gfp and the control of spicules during copulation is to identify target genes regulated by UNC-55, a putative transcription factor. We compared the mRNA populations of two him-5 strains, one homozygous for unc-55(+) and the second homozygous for unc-55 (e1170) using Representational Differentiation Analysis. We have sequenced 48 different clones representing 36 different cDNAs. The transcription of each of the cDNAs appeared to be activated by UNC-55. Attempts to identify cDNAs suppressed by UNC-55 did not yield any products. The cDNAs fell into five categories: muscle specific (4), cytoskeletal (5), protein synthesis (9) signal transduction (5) and novel (4). The remaining represented flanking fragments of products that had already been identified. Reverse northern blots will be used to confirm the differential expression of the mRNAs in the two populations. We are encouraged by the correlation between the cell types, the potential function in copulation and the categories of genes that are potentionally regulated either directly or indirectly by UNC-55 in those cell types.

Muhlrad PJ et al. (2000) Worm Breeder's Gazette "Locating spe-6 by mapping the overlap of two LGIII deficiencies, tDf7 and ctDf3"

Ward S, Muhlrad PJ

[Worm Breeder's Gazette, 2000]

In the later half of the second millennium, when we first set out to clone the spe-6 gene, the physical map in its region of the right arm of chromosome III was tattered and moth-eaten. spe-6 lay in a large hole to the right of tra-1. When nob-1 was cloned, the hole appeared patched, so we were able to gain a foothold to move in on spe-6. spe-6 is the only reported gene whose mutations fail to complement both of the overlapping deficiencies, tDf7 and ctDf3. We therefore decided to locate this overlap region on the physical map. We used PCR on individual dead homozygous ctDf3 or tDf7 mutant embryos, targeting regions of the genome that we suspected either closely flanked or fell within one or both of the deficiencies. With each tester primer set, we also multiplexed a set of primers whose target we knew fell under the deficiency and one primer set whose target was definitely outside of the deficiency. These controls proved essential, because we frequently encountered false positives and false negatives in our PCR reactions. Starting with targets in cosmid T28D6 and YAC Y41C4, we honed in on the overlap region until we had narrowed it down tooblivion. We could not identify a region on the physical map that was deleted in both deficiencies. Further molecular analysis of the region (represented in YAC Y66A7, accession # AL161712) revealed that its structure was not as depicted on the physical map. Following a series of increasingly pathetic emails to the Sanger Centre, Alan Coulson pulled out a new YAC, Y66D12, that bridged the newly discovered gap, and John Sulston (himself!) constructed a library and sequenced the YAC. The new sequence, about 166 kb, showed numerous extended repeats, which proved difficult to sequence, and were probably responsible for the problems with cloning this region. Continuing with our PCR strategy, we narrowed the tDf7/ctDf3 overlap to within about 12 kb, a region containing three tightly packed genes, represented by Kohara EST clones. EST clones from the leftmost gene are from a hermaphrodite embryonic library, suggesting that this gene was probably not spe-6, a gene involved in spermatogenesis. The other two genes both had clones from a him-8 library. Most enlightening, the middle gene was also was also represented by a clone isolated recently by Harold Smith in our laboratory from a normalized and subtracted cDNA library highly enriched for spermatogenesis genes (i.e. expressed in fem-3 (q23gf) mutant hermaphrodites, which make only sperm, but not in fem-1(hc17ts) mutants, which make only oocytes). Further analysis of this gene (described in the accompanying article) confirmed that it is in fact spe-6.