Andrew C Giles et al. (2005) International Worm Meeting "The role of dopamine in mechanosensory long-term memory"

Andrew C Giles, Catharine H Rankin, Hanna Weirich

[International Worm Meeting, 2005]

Dopamine plays an important role in the behavior of Caenorhabditis elegans, modulating mechanosensation, locomotion, feeding, and egg laying (review:1). Recently, it has been shown that dop-1 mutants, who lack a dopamine receptor, habituate to the tap withdrawal response more quickly than wild-type worms, as measured by frequency of reversals, suggesting that dopamine may play a role in mechanosensory learning in C. elegans (2). Glutamatergic neurotransmission is thought to play a role in this mechanosensory learning and the formation of long-term memory for habituation to tap (3-5). In other organisms, dopamine often modulates glutamatergic neurotransmission, such as in incentive learning in mammals (review:6), and recently, it has been shown that glutamate and dopamine interact to mediate an area restricting search behavior in C. elegans (7); therefore, we hypothesized that dopamine modulates the formation of long-term memory in C. elegans. Mutant strains with deficits in their dopaminergic system, cat-2 (tyrosine hydroxlase; received from CGC) and dop-1 (dopamine receptor; received from Van Tol), were tested for long-term memory using the long-term habituation of the tap withdrawal response protocol (4). Both mutants appear to show approximately normal short-term habituation, as measured by magnitude of reversals; however, neither strain showed evidence of long-term memory for habituation. This suggests that dopaminergic neurotransmission is important in modulating long-term memory of mechanosensory stimuli. Dopamine neurotransmission can act extrasynaptically in C. elegans (8), so perhaps dopamine works by setting an overall tone as it has been hypothesized in the nervous system of mammals in order to modulate working memory (review:9). We are presently investigating whether the memory deficit seen in cat-2 mutants can be rescued by the application of exogenous dopamine. References: 1) Nass and Blakely (2003) Annu Rev Pharmacol Toxicol 43:52144; 2) Sanyal et al. (2004) EMBO 23:47382; 3) Rankin and Wicks (2000) J Neurosci 20:433744; 4) Rose et al. (2002) Learn Mem 9:1307; 5) Rose et al. (2003) J Neurosci 23:95959; 6) Beninger and Gerdjikov (2004) Neurotox Res 6:91-104; 7) Hills et al. (2004) J Neurosci 24:121725; 8) Chase et al. (2004) Nat Neurosci 7:10961103; 9) Seamans and Yang (2004) Prog Neurobiol 74:157.

Giles, Andrew C. et al. (2013) International Worm Meeting "Toward the identification of behavioral strategies underlying C. elegans thermotaxis using the Multi-Worm Tracker."

Tsukada, Yuki, Giles, Andrew C., Mori, Ikue, Nakano, Shunji

[International Worm Meeting, 2013]

C. elegans navigate thermal gradients to find their cultivation temperature. Studying mechanisms of thermotaxis will provide insight into neural functions including thermosensation, memory and the computations that neurons use to integrate information and decide on motor output. Assays that measure the accumulation of worms on thermal gradients rapidly quantify thermotaxis but are not capable of determining the behavioral strategies that worms utilize while navigating. Analyses of locomotion on thermal gradients and in response to temperature ramps have revealed some of the underlying strategies, such as biased random walk during cryophilic movement, but have not yet identified components needed for other aspects of the behavior, including thermophilic movement. To develop a high-throughput assay that captures components of locomotion in environments where thermophilic movement has been observed, we adapted the Multi-Worm Tracker (MWT), a software-hardware package that measures locomotion-based behavior for dozens of worms simultaneously, to work with our accumulation assay. There were two main obstacles: lighting and the size of the field of view. We found that using black anodized aluminum under the assay plate to transmit the thermal gradient in combination with low angle lighting above provided good contrast between worms and background. To image a 13.5 x 10 cm thermotaxis assay plate, we found the Dalsa Falcon 4 MP camera previously used by the MWT did not provide enough resolution to adequately visualize body shape, which impaired automated identification of certain components of locomotion. We found the Toshiba-Teli CleverDragon 12 MP camera was compatible and improved the imaging of body shape and locomotion while maintaining adequate temporal resolution (~13 Hz). We conclude that this setup can be used to track locomotion components during multi-worm thermotaxis assays and can likely be applied to analyze other behaviors that require a large assay area, such as chemotaxis. We hope that high-throughput analysis with this system will reveal more of the behavioral strategies underlying C. elegans thermotaxis behavior.

Giles, Andrew C. et al. (2011) International Worm Meeting "Secondary Allele Screen for Extreme Habituation Phenotypes after High Throughput Behavioral Characterization of a Nervous-system-biased Mutant Library."

Kerr, Rex A., Swierczek, Nicholas A., Giles, Andrew C., Rankin, Catharine H.

[International Worm Meeting, 2011]

Habituation is the most simple and fundamental form of learning and is measured as a decrease in response to a repeated stimulus. We assay habituation of the tap withdrawal response in C. elegans using the Multi-Worm Tracker (MWT), which allows rapid characterization of tap habituation and therefore enables the testing of large numbers of mutants. The assay consists of 10 minutes of recording prior to the stimulation protocol to assess locomotion and spontaneous reversal rates followed by thirty mechanical taps to the side of the plate at a 10 second inter-stimulus interval. In wild-type worms, the tap initially elicits a reversal response that gradually habituates with repeated presentations. We have collected and tested a nervous-system-biased mutant library (~700 strains) by cross-referencing a list of 2073 genes with predicted neural function based on domain structure (Sieburth et al. Nature:436, 2005) with the list of available strains at the Caenorhabditis Genetics Center. This identified a number of mutants with habituation phenotypes, but it was unclear whether these phenotypes were caused by the known mutation, or background mutations. We have now screened a set of secondary (and for some, tertiary) mutations to test if the phenotypes are consistent between alleles. The genes that cause the strongest effects on habituation and were consistent across all tested alleles encode the G-alpha-protein goa-1 and the regulator of G-protein signaling (RGS) known as eat-16. We are currently following up on these genes as well as others to develop new insights into the molecular mechanism of habituation.

Fire A et al. (1994) Worm Breeder's Gazette "Vectorology I: New lacZ Vectors (Building a Better Gene Trap)."

Fire A, Xu SQ

[Worm Breeder's Gazette, 1994]

Vectorology I: New lacZ vectors ("building a better gene trap") Andrew Fire and SiQun Xu Carnegie Institution of Washington, Baltimore, Md 21210

Wendy Wong et al. (2006) European Worm Meeting "The Integrative Interaction Map for Caenorhabditis elegans"

Wendy Wong, Andrew Fraser

[European Worm Meeting, 2006]

. Wendy Wong and Andrew Fraser. C. elegans is a popular model system for the systematic analysis. However, relatively little is known for its networks of genetic interactions. We hereby construct an integrative interaction map for C. elegans by integrating diverse functional genomics Datasets. We will also present some of the novel tools in querying the interaction map for answering powerful biological hypotheses.

Mehra A et al. (1994) Worm Breeder's Gazette "Direct Interaction Between FEM-3 and a Carboxy-Terminal Fragment of TRA-2A"

Spence AM, Heck L, Kuwabara PE, Mehra A

[Worm Breeder's Gazette, 1994]

Direct Interaction between FEM-3 and a Carboxy-Terminal Fragment Or TRA-2A Arun Mehra, Linda Heck, Patricia Kuwabara*, and Andrew Spence Dept. of Medical Genetics, University of Toronto, 1 King's College Circle, Toronto, Canada, and *MRC Laboratory of Molecular Biology, Hills Rd., Cambridge, UK

Nakano, Shunji et al. (2015) International Worm Meeting "The C. elegans MAST Kinase Acts through Stomatin to Regulate Thermotaxis Behavior."

Sano, Ayana, Nakano, Shunji, Mori, Ikue, Ikeda, Muneki, Higashiyama, Tetsuya, Suzuki, Takamasa, C. Giles, Andrew

[International Worm Meeting, 2015]

C. elegans can sense and remember the environmental temperature and navigate themselves toward that temperature when placed on a thermal gradient. Although previous studies identified genes and neural circuits involved in thermotaxis, how C. elegans achieves this complex behavior still remains elusive. To further reveal the mechanisms underlying thermotaxis, we undertook a genetic screen to look for mutants defective in thermotaxis. We found that a null mutation in the gene kin-4, which encodes the C. elegans homolog of MAST (Microtubule Associated Serine Threonine) kinase, caused a cryophilic phenotype. Expression of kin-4 in the AFD thermosensory neuron rescued this defect. We also found that nj89, another isolate recovered from our screen, caused a thermophilic defect and suppressed the kin-4 mutant phenotype. nj89 is a gain-of-function mutation of mec-2, which encodes a stomatin-like membrane-associated protein previously shown to bind to microtubules and a DEG/ENaC (Degenerin/Epithelial Na Channel). To identify the behavioral components defective in these mutants, we set up a high-throughput tracking system to record thermotaxis behavior. Wild-type animals modulate the frequencies of omega and reversal turns. They also bias the angle of shallow turns to steer toward the cultivation temperature. We found that kin-4 and mec-2 mutants are defective in regulating reversal turn frequencies . Our results suggest a novel signal transduction pathway in which KIN-4/MAST kinase regulates MEC-2/Stomatin. Since both a mammalian MAST kinase and MEC-2 are known to associate with microtubules, KIN-4 might bind to microtubules and regulate the activity of MEC-2. Furthermore, given that MEC-2 modulates ENaC activity and that some ENaCs respond to temperature change, the KIN-4/MEC-2 pathway might regulate ENaC activity and control a process in the AFD sensory neuron, such as temperature sensing. Further study will assess whether ENaCs act with KIN-4/MEC-2 during thermotaxis and identify the molecular and neural mechanisms by which KIN-4/MEC-2 controls thermotaxis behavior.

Desbois M et al. (2023) STAR Protoc "Optimized protocol for in vivo affinity purification proteomics and biochemistry using C. elegans."

Desbois M, Grill B, Pak JS, Opperman KJ, Giles AC

[STAR Protoc, 2023]

We present an optimized protocol for in vivo affinity purification proteomics and biochemistry using the model organism C. elegans. We describe steps for target tagging, large-scale culture, affinity purification using a cryomill, mass spectrometry and validation of candidate binding proteins. Our approach has proven successful for identifying protein-protein interactions and signaling networks with verified functional relevance. Our protocol is also suitable for biochemical evaluation of protein-protein interactions in vivo. For complete details on the use and execution of this protocol, please refer to Crawley et al.,¹ Giles et al.,² and Desbois et al.³.

(2021) Development "The people behind the papers - Clement Dubois, Shivam Gupta, Andrew Mugler and Marie-Anne Felix."

[Development, 2021]

Cell migration needs to be precisely regulated during development so that cells stop in the right position. A new paper in Development investigates the robustness of neuroblast migration in the <i>C. elegans</i> larva in the face of both genetic and environmental variation. To hear more about the story, we met the paper's four authors: Clement Dubois and Shivam Gupta, and their respective supervisors Andrew Mugler (currently Assistant Professor at the Department of Physics and Astronomy at the University of Pittsburgh, where his lab recently moved from Purdue University) and Marie-Anne Felix (Principal Investigator at Institut de Biologie de l'Ecole Normale Superieure in Paris and Research Director at CNRS).

Van Voorhies WA (1999) Worm Breeder's Gazette "The effects of transferring on worm fertility"

Van Voorhies WA

[Worm Breeder's Gazette, 1999]

Worm fecundity is commonly assayed in many C. elegans studies. Any measurement of worm fecundity requires proper control of environmental factors that potentially affect worm fertility. In a recent article in the WBG (Twistering: A new approach to fecundity assays), Andrew Stewart mentioned that the developmental stage at which a worm is transferred significantly affected worm fecundity. Stewart reported that progeny counts recorded from worms transferred to plates as L3 worms were significantly less than progeny counts recorded from worms transferred to plates when either eggs or L2 worms. This implies that transferring older larval worms can damage the worm enough to reduce its fertility.