Tzoneva M et al. (1998) West Coast Worm Meeting "CHARACTERIZATION AND CLONING OF THE MUSCLE ACTIVATION GENE UNC-58"

Tzoneva M, Thomas JH

[West Coast Worm Meeting, 1998]

unc-58 was first identified by dominant mutations that cause hypercontracted body-wall and egg-laying muscle in C. elegans. unc-58(dm) animals are rigidly paralyzed and egg-laying constitutive. Putative loss-of-function unc-58 alleles have been isolated as revertants of the unc-58(dm) phenotype. These alleles have no obvious phenotype on their own, suggesting that unc-58(dm) mutants result in an inappropriately activated gene product. unc-58(dm) stands out among other muscle activation mutations because it has the following unusual phenotypes. First, unc-58(dm) animals frequently flip around their longitudinal axis. This phenotype is, to our knowledge, not found in any other C. elegans mutant. Second, the unc-58(dm) phenotype is partially rescued by the drug endosulfan, an antagonist of GABA-gated chloride channels (B. Wightman and G. Garriga, personal communication; our unpublished data). Third, unc-58(dm) alleles exhibit a defect in the outgrowth of HSN neuron projections (B. Wightman and G. Garriga, personal communication). We plan to clone the unc-58 gene and perform further genetic, pharmacological and molecular experiments to elucidate its mechanism and site of function. unc-58 has been mapped to a region between unc-115 and dpy-6 on the X chromosome (D. Thierry-Mieg, personal communication). We have obtained putative Tc1 insertions in unc-58 by screening in a mut-6 background for revertants of the uncoordinated phenotype of an unc-58(dm) allele. We will use cosmids that cover the unc-58 region as probes to identify the Tc1 insertions. Because the unc-58(dm) mutation affects muscle excitability, it may encode an ion channel or a regulator of ion channel activity.

M Tzoneva et al. (1999) International C. elegans Meeting "Characterization and cloning of the muscle activation gene unc-58"

M Tzoneva, JH Thomas

[International C. elegans Meeting, 1999]

unc-58 was first identified by dominant mutations that cause hypercontracted body-wall and egg-laying muscle in C. elegans . unc-58(dm) animals are rigidly paralyzed and egg-laying constitutive. Putative loss-of-function unc-58 alleles have been isolated as revertants of the unc-58(dm) phenotype. These alleles have no obvious phenotype on their own, suggesting that unc-58(dm) mutants result in an inappropriately activated gene product. unc-58(dm) stands out among other muscle activation mutations because it has the following unusual phenotypes. First, unc-58(dm) animals frequently flip around their longitudinal axis. This phenotype is, to our knowledge, not found in any other C. elegans mutant. Second, the unc-58(dm) phenotype is partially rescued by the drug endosulfan, an antagonist of GABA-gated chloride channels (B. Wightman and G. Garriga, personal communication; our unpublished data). Third, unc-58(dm) alleles exhibit a defect in the outgrowth of HSN neuron processes (B. Wightman and G. Garriga, personal communication). Because the unc-58(dm) mutation affects muscle excitability, it may encode an ion channel or a regulator of ion channel activity. We plan to clone the unc-58 gene and perform further genetic, pharmacological and molecular experiments to elucidate its mechanism and site of function. We have mapped unc-58 to a small region just to the left of unc-115 . We have obtained putative Tc1 insertions in unc-58 by screening in a mut-6 background for revertants of the uncoordinated phenotype of an unc-58(dm) allele. We are now using cosmids from the unc-58 region as probes to identify the Tc1 insertions.

Fragkiadaki, Persefoni et al. (2021) International Worm Meeting "Insecticide resistance and toxicity mechanisms in malaria vectors and C. elegans"

Fragkiadaki, Persefoni, Lionaki, Eirini, Tavernarakis, Nektarios, Balabanidou, Vasileia, Vontas, John, Kounakis, Kostas, Charamis, Iason, Kefi, Mary

[International Worm Meeting, 2021]

Malaria continues to claim more than 400.000 lives every year, and represents one of the biggest health issues for humanity worldwide. Deltamethrin (DM) is one of the most potent insecticides used to eradicate malaria and belongs to type II pyrethroids. DM targets voltage-gated sodium channels (VGSCs) expressed in neuronal cells of target species. However, the off-target effects of chronic exposure to DM are poorly characterized. We use C. elegans as a non-target species, and the malaria vector Anopheles gambie, to identify molecular pathways that mediate insecticide toxicity and/or resistance to DM. Proteomic analysis of resistant mosquitoes compared to susceptible counterparts shows increased levels of OXPHOS and proteasomal proteins, and decreased levels of aminoacyl-tRNA biosynthesis and ribosomal proteins, suggesting reduction in protein translation. The proteomic profile of DM resistant mosquitoes resembles the profile of low insulin/IGF1 signaling (IIS) C. elegans mutants and indicates that resistant mosquitoes could experience low IIS. We tested the effects of DM on nematodes treated throughout their post-embryonic development. DM treated worms can develop to adulthood and produce viable progeny. We found that DM does not induce sod-3 expression in wt animals, rather it attenuates sod-3 induction in daf-2 mutants and starved wt animals. In addition, DM significantly induces ER stress and antioxidant response in a dose dependent manner, while it alters the exploratory behavior of wild type worms in the presence of food. Our findings implicate IIS in deltamethrin-induced toxicity, in non-target species and in the development of mosquito resistance mechanisms. We are currently investigating whether modulation of IIS could serve as a putative toxicity and resistance management strategy.

Wang, C. et al. (2019) International Worm Meeting "Expression and functional studies of the DM-domain transcription factors reveal novel sexual dimorphisms."

WANG, C., Hobert, O.

[International Worm Meeting, 2019]

Understanding structural and functional differences in the brains of females and males is fundamental for explaining sexually dimorphic animal behaviors. Despite variations in sexual differentiation pathways among species, a common theme adapted across phyla is the use of the DM (Drosophila doublesex/C. elegans mab-3 domain) family transcription factors, making them ideal candidate genes for studying mechanisms behind sexual dimorphisms. The DM family genes are expanded extensively in various species and have been implicated in regulating the development of somatic gonads and differentiation of neurons. The C. elegans genome encodes 11 DM genes, two of which conserved in humans. Only a small subset of these genes have been characterized in detail before. By CRISPR/Cas9-mediated gfp tagging of all 11 endogenous DM genes, we discovered novel sexual dimorphisms in the C. elegans nervous system. In contrast to previously reported sexually dimorphic expression of DM genes in a few sensory neurons mostly born only in males and not hermaphrodites (somatic females), our CRISPR lines reveal that of the 294 neurons that are lineally and anatomically generated in both sexes (sex-shared), about 40% of them (or 24% of sex-shared neuron classes) express a DM gene exclusively in males. Importantly, these dimorphic neurons include not only sensory, but also inter- and motor neurons. Further, based on known connectome studies, some of these neurons generate sexually dimorphic synaptic connections with other sex-shared neurons, indicating potential roles of DM genes in regulating sexual dimorphisms on a synaptic level. Mutant analysis showed that mab-3, one of the DM genes, contributes to sexually dimorphic locomotory behaviors. Taken together, completion of this work using the conserved DM genes for studying neuronal sexual dimorphisms will shed light on our understanding on sex differences in the nervous system.

Kostich M et al. (1998) East Coast Worm Meeting "Molecular-Genetic Analysis of a LAMP-like Gene from C. elegans"

Fambrough DM, Fire A, Kostich M

[East Coast Worm Meeting, 1998]

Members of the lysosome associated membrane protein (LAMP) family are abundantly expressed in all vertebrate cells that have been examined. They are found predominantly in lysosomes, and in lesser amounts in endosomes and at the cell surface. The function of LAMPs is not known, but they have been implicated in normal cellular adhesion, as well as in the process of metastatic invasion. The sensitive search algorithm of Gribskov et al. (Methods in Enzymology, 183:146-59, 1989) was used to search publicly available sequence databases for invertebrate genes potentially related to the vertebrate LAMP family. A candidate was found in genomic sequence from C. elegans. Analysis of expressed sequence tag (EST) data revealed the existence of corresponding full length C. elegans cDNAs and confirmed the transcript structure predicted for this gene. Partial sequences very similar to corresponding regions of the C. elegans gene were found in EST data from the nematodes Brugia malayi and Pristionchus pacificus. Although little is known about LAMP function, protein segments responsible for lysosomal sorting have been delineated. In order to estimate the similarity of this targeting signal between man and worm, the nematode cDNA was modified to code for an epitope tagged form of the worm protein, and expressed in cultured mammalian cells. The tagged nematode protein was targeted to lysosomes in these cells, and site directed mutation was used to show the involvement of a motif nearly identical to that involved in sorting of vertebrate LAMPs to lysosomes. In order to investigate the expression pattern of the worm gene, genomic upstream sequence was used to drive GFP expression in developing animals, and in situ hybridization to mRNA was performed (special thanks to Brian Harfe for performing the hybridization). Both types of experiments indicated nearly ubiquitous transcription of this gene. We also raised polyclonal antiserum against the worm protein. Staining of whole worms with this serum revealed strong staining in a subset of gut granules in embryos, larvae, and adults. A mutant allele in which the first three exons are deleted, was kindly provided by NemaPharm (Cambridge, MA). We found that this mutant fails to produce detectable levels of protein, is homozygous viable, grows at the normal rate, and has a slightly reduced brood size. The guts of well fed mutant worms contain fewer gut granules than normal, but worm lipofuscin deposition, gut granule staining with acridine orange, and uptake of labeled dextran seem normal. We are further characterizing the mutant at the physiological and ultra structural level.

Monika Tzoneva et al. (2001) International C. elegans Meeting "UNC-58 IS AN UNUSUAL POTASSIUM CHANNEL OF THE TWIK FAMILY"

James H Thomas, Monika Tzoneva

[International C. elegans Meeting, 2001]

unc-58 was first identified by dominant mutations that cause hypercontracted body-wall and egg-laying muscle in C. elegans . unc-58(dm) animals are rigidly paralyzed and egg-laying constitutive. unc-58(dm) animals also frequently flip around their longitudinal axis. Putative loss-of-function unc-58 alleles have been isolated as revertants of the unc-58(dm) phenotype. These alleles have a much milder and distinct phenotype, suggesting that unc-58(dm) mutants result in an inappropriately activated gene product. We have cloned unc-58 and found that it encodes a member of the TWIK potassium channel family. TWIKs are a distinct family of potassium channels having four transmembrane domains (M1 to M4) and two pore domains. Potassium-selective currents have been recorded from TWIKs from both mammals and worms. The hypercontracted phenotype of unc-58(dm) is in sharp contrast to that of all other activated potassium channel mutants, which lead to muscle relaxation caused by excessive inhibitory currents. Two scenarios can explain this hypercontraction. First, UNC-58(dm) may produce excessive potassium currents in the inhibitory motorneurons. However, this does not account for the hypercontraction of the egg-laying muscle, which is not innervated by inhibitory motorneurons. Second,, UNC-58(dm) may act in excitatory neurons and/or muscle to conduct an excitatory current. A precedent for that is the Ih channel from the human heart, which is related to potassium channels by sequence, but is permeable to both potassium and sodium [Ludwig et al, Nature 393, 587-591]. To distinguish between these two possibilities, we are expressing UNC-58(dm) in Xenopus laevis oocytes to determine its ion selectivity and using GFP fusions to determine the UNC-58 site of action. Both the flipping and the hypercontarction phenotypes of unc-58(dm) are partially rescued by the drug endosulfan, best known as an antagonist of GABA-gated chloride channels (B. Wightman and G. Garriga, personal communication; our unpublished data). This rescue is allele specific, and is not affected by mutants in GABA-ergic neurotransmission. We plan to use electrophysiology to determine whether this rescue is by direct channel block.

Davis MW et al. (1995) International C. elegans Meeting "Mutations in the Na, K-ATPase a-subunit gene eat-6 disrupt excitable cell function"

Lee RYN, Somerville D, Fambrough DM, Davis MW, Avery L

[International C. elegans Meeting, 1995]

We have cloned a Na, K-ATPase a-subunit and shown that eat-6 mutants can be rescued by a genomic fragment containing the ATPase coding region plus 1kb of non-coding sequence on each side of the ORF. Further, eat-6 pharynxes are hypersensitive to a Na,K ATPase inhibitor and this inhibitor phenocopies eat-6 when it is applied to wild-type pharynxes. These results together with the fact that the physical map position of the ATPase gene is consistent with the interpolated position of eat-6 suggest that eat-6 encodes this ATPase. eat-6 mutations cause slow, delayed relaxations of pharyngeal muscle. These defects are due to defects in the electrical properties of the muscle because currents generated when pharynxes from eat-6 worms relax are severely reduced, as we have shown using an extracellular electrical recording technique called the electropharyngeogram. Also, the eat-6 phenotype remains when 19/20 of the pharyngeal neurons are ablated, suggesting that the phenotype is not due entirely to a nervous system defect. Using intracellular recording, we have shown that the resting membrane potential of eat-6 mutant pharyngeal muscles is consistently more depolarized compared to wild-type (-20mV to -45mV in eat-6 compared to -40mV to -60mV in wild-type). Further, eat-6 action potentials are smaller, and the return to resting potential is slower. To explain these abnormalities, we propose that a reduction of Na, K-ATPase activity in eat-6 mutants leads to a reduction of the resting potential of the cell. This reduced potential would change the gating kinetics of many voltage dependent ion channels, such as a voltage gated K+ channel which con- tributes to repolarization at the end of the action potential (Beyery and Masuda, J. Physiol. 288, 263-284).

Tzoneva M et al. (2000) West Coast Worm Meeting "Characterization and cloning of the muscle activation gene unc-58"

Tzoneva M, Thomas JH

[West Coast Worm Meeting, 2000]

unc-58 was first identified by dominant mutations that cause hypercontracted body-wall and egg-laying muscle in C. elegans. unc-58(dm) animals are rigidly paralyzed and egg-laying constitutive. Putative loss-of-function unc-58 alleles have been isolated as revertants of the unc-58(dm) phenotype. These alleles have no obvious phenotype on their own, suggesting that unc-58(dm) mutants result in an inappropriately activated gene product. unc-58(dm) animals also frequently flip around their longitudinal axis. Both the flipping and the hypercontarction phenotypes are partially rescued by the drug endosulfan, best known as an antagonist of GABA-gated chloride channels (B. Wightman and G. Garriga, personal communication; our unpublished data). I have cloned unc-58 and found that it encodes a potassium channel of the TWIK family. TWIKs are distinct from other potassium channels because they have four transmembrane domains (M1 to M4) and two pore domains. Potassium-selective currents have been recorded from TWIKs in both mammals and worms. The three unc-58 gain-of-function mutations cluster in the C-terminal (cytoplasmic) part of the M4 transmembrane domain, which is equivalent to the S6 domain of the voltage-gated potassium channel family. S6 is thought to form a channel gate that opens in response to voltage changes. The location of the unc-58(dm) mutations suggests that its M4 helix may also form an activation gate, though the stimulus that opens it is unknown. The molecular identity of unc-58 raises several questions. First, how does an activated mutation in a putative potassium channel lead to muscle hypercontraction? It is possible that the hypercontraction is due to UNC-58 function in inhibitory motorneurons, while its slow movement may be due in part to its function in other tissues. We will test this by determining the unc-58 site(s) of action. Second, how is the unc-58(dm) phenotype rescued by the drug endosulfan? We will attempt to determine whether this rescue is by direct channel block. Third, how does the UNC-58 channel contribute to overall membrane excitability? It is not known whether or how UNC-58 activity is regulated and whether it acts alone or in complex with other subunits. We have isolated extragenic suppressors of unc-58(dm), which may offer insight into this question.

Davis MW et al. (1996) Midwest Worm Meeting "EAT-6 ENCODES A NA+/K+ ATPASE a-SUBUNIT"

Fambrough DM, Sommerville D, Avery L, Lee RYN, Davis MW, Lockery SR

[Midwest Worm Meeting, 1996]

We have cloned a Na+/K+ ATPase a-subunit gene and shown that it is encoded by the gene eat-6. eat-6 mutant worms are feeding defective. Their pharyngeal muscles have feeble contractions and slow, delayed relaxations. The electropharyngeogram (EPG) currents associated with muscle relaxation are reduced in eat-6 worms, demonstrating that the mutants have a defect in the pharyngeal muscle action potential. The magnitude of this defect correlates with the degree of starvation seen in a series of four eat-6 alleles and in one cold-sensitive allele raised at different temperatures. Ouabain, a sodium pump inhibitor, phenocopies the eat-6 electrical defect in isolated wild-type pharynxes, and pharynxes from eat-6 worms are hypersensitive to the drug. In order to examine the electrical defect of eat-6 in more detail, we developed an intracellular recording technique for the pharyngeal muscles. With this, we found that wild-type pharynxes have a resting membrane potential around -45mV, an action potential peak near 34mV and an overshoot at the end of the action potential to around -67mV. We found that eat-6 pharynxes have a more positive resting potential (-35mV), a reduced action potential amplitude (15mV) and a reduced overshoot (-49mV). In addition, the repolarization phase of eat-6 action potentials are slower. These electrical defects are consistent with the defects seen with the EPG records. This slower repolarization phase also explains the slow, delayed relaxations seen in eat-6 pharynxes. We propose that the electrical changes seen in eat-6 pharynxes can be explained by two effects. First, the mutations cause a reduction in voltage across the muscle membrane. Second, this voltage change alters the kinetics of voltage dependent ion channels, leading to both changes in magnitude and timing of phases of the action potential.

Monika Tzoneva et al. (2004) West Coast Worm Meeting "unc-58 encodes a member of the TWIK potassium channel family which may have altered selectivity for potassium"

Monika Tzoneva, James H Thomas

[West Coast Worm Meeting, 2004]

Dominant, gain-of-function mutations in the C. elegans unc-58 gene have hypercontracted body-wall and egg-laying muscle and are severely uncoordinated, while loss-of-function mutations have no observable phenotype. I have cloned unc-58 and found that it encodes a member of the TWIK (two-pore domain) potassium channel family. The unc-58 dominant mutations cluster in the fourth transmembrane domain (the M4). This location suggests that the UNC-58 M4 helix may form an activation gate, which opens the channel in response to an unknown stimulus. The insecticide endosulfan, best known as a GABA-A receptor antagonist, rescues the phenotypes of the unc-58(e665dm) and unc-58(e757dm) alleles completely, while the unc-58(n495dm) allele is not affected by endosulfan treatment. Our experiments suggest that endosulfan acts directly and specifically to block the UNC-58 channel. How does an activated mutation in an inhibitory channel lead to muscle hypercontraction? It is possible that that the unc-58(dm) hypercontraction phenotype may be due to its function in inhibitory motorneurons. Alternatively, UNC-58 may act in excitatory neurons and/or muscle in order to conduct an excitatory current such as sodium. We show that unc-58 is expressed in the excitatory motorneurons and head neurons, and that mutations eliminating inhibitory GABA-ergic neurotransmission do not suppress the unc-58(dm) phenotypes. These results suggest that the activated UNC-58 channel may have relaxed selectivity for potassium. In order to identify molecules interacting with the UNC-58 channel, we have isolated multiple unc-58(dm) extragenic suppressor alleles. These fall into at least three complementation groups and two phenotypic classes. Different aspects of the unc-58(dm) phenotype are specifically suppressed by mutants from the two classes, indicating that suppression may be tissue-specific or specific to a certain aspect of UNC-58 function.