Jungsoo Lee et al. (2007) International Worm Meeting "Chronic effects of Ethanol in C. elegans."

Steven L. McIntire, Jungsoo Lee

[International Worm Meeting, 2007]

Ethanol can affect multiple signaling pathways. Some of these pathways have been implicated in acute or chronic behavioral responses to ethanol (Diamond and Gordon, 1997). Ethanol alters the behavioral responses of C. elegans at the same tissue concentrations that cause intoxication in humans (Davies et al., 2003). Similar molecular pathways appear to contribute to the acute neurodepressive effects of ethanol that are observed in C. elegans and mammalian systems. Ethanol exposure during development causes a variety of morphological changes and a retardation of growth. We are interested in identifying the molecular pathways that contribute to the developmental and behavioral defects that are observed after chronic ethanol exposure. We have observed that ethanol inhibits the growth of wild-type animals. To better understand the mechanisms contributing to this effect, we carried out screens for mutants resistant to the chronic effects of ethanol. We have screened 5,000 haploid genomes and have isolated 14 candidates showing resistance to ethanol inhibition of growth. Several mutants are also resistant to acute effects of ethanol on locomotion and egg laying. Some of the mutants have a defect in the acute tolerance that occurs during a continuous ethanol exposure. None of the mutants have an obvious behavioral phenotype in the absence of ethanol. One possibility is that chronic ethanol induces oxidative stress by enhancing reactive oxygen species (ROS) formation or by decreasing oxidative defenses (Sun et al., 2001). To assess the relationship between resistance to ethanol and oxidative stress, we examined sensitivity of the mutants to paraquat. A subset of the mutants is also resistant to paraquat. We have mapped two mutants to the center of chromosome X and are currently in the process of cloning these genes. Reference: Diamond I and Gordon AS (1997). Cellular and molecular neuroscience of alcoholism. Physiol Rev. 77(1):1-20. Review Davies AG et al. (2003). A central role of the BK potassium channel in behavioral responses to ethanol in C. elegans. Cell 115(6):655-66.

Jungsoo Lee et al. (2003) International Worm Meeting "Characterization of a gain-of-function mutant of Calcineurin A (cna-1)"

Jungsoo Lee, Changhoon Jee, Joohong Ahnn

[International Worm Meeting, 2003]

Calcineurin is a calcium-calmodulin dependent protein phosphatase that is a key component of Ca2+-related signaling pathways. Calcineurin functions as a heterodimer, consisting of a catalytic subunit (CnA) and a regulatory subunit (CnB). This two-subunit structure is well conserved from yeast to human. Previously, we have isolated and characterized a null mutant of calcineurin B [cnb-1 (jh103)] which showed the severe defects in locomotion, egg laying and displayed serotonin-mediated egg-laying resistance.1 Interestingly, very similar phenotypes were also observed in gain-of-function mutant of unc-43, which encodes the calcium-calmodulin dependent protein kinase CaMKII.2 Taken together, it has been suggested that cnb-1 and unc-43are involved in the G-protein signaling pathway which regulates locomotion and egg laying. Recently, we have isolated a gain-of-function mutant of catalytic subunit, calcineurin A [cna-1 (jh107)] which is deleted in the calmodulin binding site and the autoinhibitory region of this phosphatase. cna-1 (jh107) shows hyperactive serotonin-mediated egg laying which is opposite phenotypes of cnb-1 (jh103). Genetic interaction between cna-1 other mutants supports the fact that calcineurin is indeed a component of G-protein signaling pathway and is involved in locomotion and egg laying in C. elegans. 1. Mol Biol Cell. 2002 Sep;13(9):3281-93. 2. Genetics. 2000 Nov;156(3):1069-82.

Jungsoo Lee et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Chronic Effects of Ethanol in C. elegans"

Jungsoo Lee, Steven McIntire

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

Ethanol can affect multiple signaling pathways. Some of these pathways have been implicated in acute or chronic behavioral responses to ethanol (Diamond and Gordon, 1997). Ethanol alters the behavioral responses of C. elegans at the same tissue concentrations that cause intoxication in humans (Davies et al., 2003). Similar molecular pathways appear to contribute to the acute neurodepressive effects of ethanol that are observed in C. elegans and mammalian systems. Ethanol exposure during development causes a variety of morphological changes and a retardation of growth. We are interested in identifying the molecular pathways that contribute to the developmental and behavioral defects that are observed after chronic ethanol exposure. We have observed that ethanol inhibits the growth of wild-type animals. To better understand the mechanisms contributing to this effect, we carried out screens for mutants resistant to the chronic effects of ethanol. We have screened 5,000 haploid genomes and have isolated 14 candidates showing resistance to ethanol inhibition of growth. Some of the mutants are also resistant to acute effects of ethanol on locomotion and egg laying. None of these mutants has an obvious behavioral phenotype in the absence of ethanol. One possibility is that chronic ethanol induces oxidative stress by enhancing reactive oxygen species (ROS) formation or by decreasing oxidative defenses (Sun et al., 2001). To assess the relationship between resistance to ethanol and oxidative stress, we examined sensitivity of the mutants to paraquat. A subset of the mutants is also resistant to paraquat. We are currently in the process of mapping and cloning some of the genes responsible for these phenotypes.

Jungsoo Lee et al. (2001) International C. elegans Meeting "Identification and characterization of a Ubiquitin C-terminal Hydrolase in C. elegans"

Joohong Ahnn, Jungsoo Lee

[International C. elegans Meeting, 2001]

Ubiquitin, a highly conserved protein composed of 76 amino acids, is important for many cellular processes including the regulation of intracellular protein breakdown, cell cycle regulation, and stress response. Ubiquitin C-terminal hydrolase (UCH) is a thiol protease that recognizes and hydrolyzes the peptide bond at the C- terminal glycine of ubiquitin. This enzyme is involved in the processing of poly-ubiquitin precursors as well as ubiquitinated proteins. UCHs have two homologous regions; the first region contains a conserved cysteine domain, the second contains two conserved residues. These two homologous regions are likely implicated in the catalytic mechanism. We searched the worm genome database and located a putative homologue of UCH in the cosmid ZK328 (LGIII). C. elegans UCH (ZK328.1) consists of 14 exons encoding 1178 amino acids and contains two functional domains that are well conserved in other organisms. Expression pattern was investigated using GFP and is shown to express in excretory cells, coelomocytes, hypodermal cells, pharynx, and neuronal cells. Northern blot experiments revealed an mRNA transcript of 3.7 kb. To better understand the function of UCH in C. elegans , RNA-mediated interference (RNAi) technology was conducted and resulted in high embryonic lethality suggesting that Ce-uch-1 has an essential role in the early embryo development of C. elegans . The RNAi phenotype was rescued by mating with wild type males. Furthermore, whole mount immunostaining showed that UCH stains the microtubule organizing center (MTOC) in fertilized embryo and in the sperm cytosol. Our data suggests that UCH is indispensable for the formation of a sperm-contributed functional MTOC and early cleavage during embryogenesis in C. elegans .

Jungsoo Lee et al. (2004) West Coast Worm Meeting "Opposing Functions of Calcineurin and CaMKII Regulate G-protein Signaling in Egg-laying Behavior of C. elegans"

Jungsoo Lee, Joohong Ahnn

[West Coast Worm Meeting, 2004]

Ca 2+ /calmodulin-dependent calcineurin has been shown to have important roles in various Ca 2+ signaling pathways. We have previously reported that cnb-1 mutants, null mutants of a regulatory B subunit, displayed pleiotropic defects including uncoordinated movement and delayed egg laying in C. elegans . Interestingly, gain-of-function mutants of a catalytic A subunit showed exactly opposite phenotypes to those of cnb-1 mutants providing an excellent genetic model to define calcium-mediated signaling pathway at the organism level. Furthermore, calcineurin is also important for normal cuticle formation, which is required for maintenance of normal body size in C. elegans . Genetic interactions between cna-1 and several mutants including egl-30 and egl-10 which are known to be involved in G-protein signaling pathway suggest that calcineurin indeed regulates locomotion and serotonin-mediated egg laying through goa-1(Go alpha ) and egl-30(Gq alpha ) . Our results indicate that, along with CaMKII, calcineurin regulates G-protein-coupled phosphorylation signaling pathways in C. elegans .

Abate JP et al. (2009) Cell Metab "Life is short, if sweet."

Blackwell TK, Abate JP

[Cell Metab, 2009]

Insulin is essential for glucose homeostasis, but reducing its activity delays the aging process in model organisms. In this issue of Cell Metabolism, Lee et al. (2009) show how these effects of insulin signaling intersect when glucose is fed to C. elegans.

Lee C et al. (2017) Bio Protoc "Single-molecule RNA Fluorescence in situ Hybridization (smFISH) in Caenorhabditis elegans."

Sorensen EB, Lee C, Seidel HS, Lynch TR, Crittenden SL, Kimble J

[Bio Protoc, 2017]

Single-molecule RNA fluorescence <i>in situ</i> hybridization (smFISH) is a technique to visualize individual RNA molecules using multiple fluorescently-labeled oligonucleotide probes specific to the target RNA ( Raj <i>et al.</i>, 2008 ; Lee <i>et al.</i>, 2016a ). We adapted this technique to visualize RNAs in the <i>C. elegans</i> whole adult worm or its germline, which enabled simultaneous recording of nascent transcripts at active transcription sites and mature mRNAs in the cytoplasm ( Lee <i>et al.</i>, 2013 and 2016b). Here we describe each step of the smFISH procedure, reagents, and microscope settings optimized for <i>C. elegans</i> extruded gonads.

Millet, Jonathan et al. (2021) International Worm Meeting "C. elegans PEZO-1 is a Mechanosensitive Channel Involved in Food Sensation"

Lee, Jungsoo, Vasquez, Valeria, Millet, Jonathan, Romero, Luis

[International Worm Meeting, 2021]

PIEZO channels are force sensors essential for physiological processes including baroreception and proprioception. The Caenorhabditis elegans genome encodes an ortholog gene of the Piezo family, pezo-1, expressed in several tissues including the pharynx. This myogenic pump is an essential component of the alimentary canal in most animals whose contraction and relaxation depends on mechanical stimulation elicited by food content. Whether pezo-1 encodes a mechanosensitive channel and contributes to pharyngeal function remains unknown. Here, we leverage genetics, microfluidics, and electropharyngeogram recordings to establish that pezo-1 is expressed in the pharynx, including a proprioceptive-like neuron, and regulates pharyngeal function. Knockout and gain-of-function mutants reveal that pezo-1 is involved in fine-tuning pharyngeal pumping frequency, sensing osmolarity and food quality. Patch-clamp electrophysiology analyses in primary C. elegans embryo cultures demonstrate that pezo-1 encodes a mechanosensitive channel. Our findings reveal a novel role for pezo-1 in regulating food sensation in worms.

Jee, Changhoon et al. (2011) International Worm Meeting "CRF receptor-like GPCR mediated stress response and regulates behavioral states in C. elegans."

Lee, Jungsoo, Garcia, L. Rene, Jee, Changhoon, McIntire, Steven

[International Worm Meeting, 2011]

Motivation is a goal directed driving force based on physiological needs such as hunger, sexual arousal and drug craving. Neuroadaptive changes in brain reward and stress system of animals leads to compulsive behaviors such as binge eating, obsessive drug seeking and sexual behavior. Repeated consumptions of drug leads to neuroadaptive changes to balance the effect of it resulted in tolerance and withdrawal symptom. We isolated SEB-3, CRF receptor-like GPCR, in C. elegans that is a key mediator in stress response. Activation of brain stress system induces behavioral arousal and development of negative reinforcement mechanism that leads to powerful motivation. We found that CRF signaling positively regulates stress response that leads to enhanced active state of locomotion, behavioral arousal and alcohol tolerance and withdrawal behavior in C. elegans. To further understand how seb-3 molecularly regulates the motivated behavioral state, we studied sex-specific mating behavior of C. elegans. The seb-3 is expressed mainly in nervous system and male sex muscles. Normally, mating potency declines in aging wild-type old males and food deprivation stress during early adulthood leads to neuroadaptive changes resulting in maintaining potency in older animals. Interestingly, food deprivation could not enhance mating potency in aging seb-3 (lf) mutant animals. Additionally, we found that seb-3 (gf) enhanced potency in old males without food deprivation and did not increase life span, suggesting that CRF signaling mediates maintaining male mating behavior of old male after food deprivation in C. elegans. We are currently pursuing an answer of where and how SEB-3 regulates cellular excitability of behavioral circuit components.

Dawes-Hoang RE et al. (2003) Dev Cell "Bringing classical embryology to C elegans gastrulation."

Zallen JA, Wieschaus EF, Dawes-Hoang RE

[Dev Cell, 2003]

In a recent paper, Lee and Goldstein develop an explant assay that recapitulates key aspects of gastrulation in C. elegans and permits classical embryological manipulations. The resulting detailed analysis of cell behavior will ultimately extend to broader issues, such as, whether morphogenesis can be described as the sum of single-cell events or if unique phenomena emerge at the multicellular level.