Slice LW et al. (1989) International C. elegans Meeting "isolation and characterization of cadmium-induced metallothionein-like protein and cDNA from C elegans."

Slice LW, Freedman JH, Rubin CS

[International C. elegans Meeting, 1989]

Slice LW et al. (1990) J Biol Chem "Purification, characterization, and cDNA cloning of a novel metallothionein-like, cadmium-binding protein from Caenorhabditis elegans."

Slice LW, Rubin CS, Freedman JH

[J Biol Chem, 1990]

Caenorhabditis elegans adapted for survival in high concentrations of Cd(II) express a heavy metal binding protein designated C. elegans metallothionein-like protein or MT-Ce. This protein was purified to homogeneity and characterized. MT-Ce binds 6 mol of Cd(II)/mol protein. The sequence of 39 amino-terminal residues in MT-Ce was determined. A radiolabeled 41-mer oligonucleotide, designed from the partial MT-Ce sequence, was used in conjunction with sucrose gradient centrifugation to obtain size-fractionated poly(A+) RNA enriched in MT-Ce sequences. Subsequently, cloned cDNAs, corresponding to MT-Ce mRNA sequences, were isolated from a lambda ZapII cDNA library prepared from the enriched template mRNA. cDNA and protein sequence analysis revealed that MT-Ce comprises 62 amino acid residues and has a predicted Mr of 6462. Seventeen of the 18 Cys residues in the nematode cadmium-binding protein are included in Cys-X-Cys and X-Cys-Cys-X motifs that are characteristic of mammalian metallothioneins (MTs). However, the resemblance of MT-Ce to mammalian MTs is superficial. The amino acid sequence of MT-Ce is unique, and neither its putative alpha and beta domains nor its Cys residues can be readily aligned with the corresponding regions of other eukaryotic MTs. This suggests that MT-Ce is an example of convergent evolution. The MT-Ce mRNA level in nematodes that were selected and grown with Cd(II) concentrations that are lethal for wild-type worms, was 55-fold higher than the level of MT-Ce mRNA in wild-type C. elegans. Comparison of the sequences of MT-Ce cDNAs revealed the occurrence of two types of MT-Ce mRNA. Each contains an identical coding region, but the cDNAs diverge markedly in their 5'-untranslated regions. This suggests the possibilities of regulation by alternative splicing and/or the presence of multiple MT-Ce genes encoding a single protein, but controlled by different regulatory elements.

Lyndal-Murphy M et al. (2010) Vet Parasitol "Reduced efficacy of macrocyclic lactone treatments in controlling gastrointestinal nematode infections of weaner dairy calves in subtropical eastern Australia."

James PJ, Pepper PM, Ehrlich WK, Rogers D, Lyndal-Murphy M

[Vet Parasitol, 2010]

Faecal Egg Count Reduction Tests (FECRTs) for macrocyclic lactone (ML) and levamisole (LEV) drenches were conducted on two dairy farms in the subtropical, summer rainfall region of eastern Australia to determine if anthelmintic failure contributed to severe gastrointestinal nematode infections observed in weaner calves. Subtropical Cooperia spp. were the dominant nematodes on both farms although significant numbers of Haemonchus placei were also present on Farm 2. On Farm 1, moxidectin pour-on (MXD) drenched at 0.5mg kg(-1) liveweight (LW) reduced the overall Cooperia burden by 82% (95% confidence limits, 37-95%) at day 7 post-drench. As worm burdens increased rapidly in younger animals in the control group (n=4), levamisole was used as a salvage drench and these calves withdrawn from the trial on animal welfare grounds after sample collection at day 7. Levamisole (LEV) dosed at 6.8mg kg(-1)LW reduced the worm burden in these calves by 100%, 7 days after drenching. On Farm 2, MXD given at 0.5mg kg(-1)LW reduced the faecal worm egg count of cooperioids at day 8 by 96% (71-99%), ivermectin oral (IVM) at 0.2mg kg(-1)LW by 1.6% (-224 to 70%) and LEV oral at 7.1mg kg(-1)LW by 100%. For H. placei the reductions were 98% (85-99.7%) for MXD, 0.7% (-226 to 70%) for IVM and 100% for LEV. This is the first report in Australia of the failure of macrocyclic lactone treatments to control subtropical Cooperia spp. and suspected failure to control H. placei in cattle.

Freedman JH et al. (1993) J Biol Chem "The novel metallothionein genes of Caenorhabditis elegans. Structural organization and inducible, cell-specific expression."

Rubin CS, Freedman JH, Slice LW, Fire A, Dixon DK

[J Biol Chem, 1993]

Two genes (mtl-1 and mtl-2) that encode the novel metallothioneins (MTs) of Caenorhabditis elegans (CeMTs) were cloned and characterized. Both genes contain a single intron that interrupts codon 6 and short 3'-untranslated regions. However, their promotor regions are distinctively non-homologous. The mtl-2 promoter contains a TATAA box and a single putative metal regulatory element. These elements are absent in the mtl-1 promoter. Nevertheless, both CeMT1 and CeMT2 mRNAs are induced by cadmium and contain precisely initiated, 5'-untranslated sequences. The inducibility and cell type specificity of metallothionein gene expression were investigated in transgenic C. elegans that carry the lacZ (beta-galactosidase) reporter gene under the control of an mtl-1 or mtl-2 promoter sequence. Upon treatment of transgenic C. elegans with cadmium or heat stress, the mtl-2:lacZ fusion gene is abundantly and exclusively expressed in the intestinal cells of larvae and adult animals. Expression is not detected in the absence of metal or heat shock. In contrast, an mtl-1:lacZ construct is constitutively expressed in the pharynx and induced by cadmium and heat shock in the intestinal cells of C. elegans larvae. The metal-inducible expression of the mtl-1:lacZ gene is attenuated in adult transgenic nematodes. Thus, the activity of each mtl promoter is modulated by metals as well as developmental and environmental factors.

Freedman JH et al. (1991) International C. elegans Meeting "MOLECULAR CHARACTERIZATION OF HEAVY METAL STRESS IN C. ELEGANS"

Linder B, Fire A, Slice LW, Dixon DK, Freedman JH, Rubin CS

[International C. elegans Meeting, 1991]

Caenorhabditis elegans provides a model system for investigating developmental and molecular mechanisms underlying adaptations to environmental stresses. We examined C. elegans adapted for growth in toxic concentrations (> 0.2mM) of Cd. Physiological responses to this persistent stress were characterized by studying the properties and expression of metallothioneins (MTs) and a group of stress-induced proteins (SIPs) that accumulate in Cd-treated nematodes. C elegans showed elevated expression of two iso-MTs, MT-l (7.9 kDa) and MT-2 (6. 4 kDa), that are 65% identical, but have no significant overall sequence homology with the conserved MTs from other species. The gene encoding MT-2 (designated mtl-2) comprises 330 bp and contains a single intron. The DNA flanking the 5'-end of mtl-2 has putative consensus sequences for a cAMP response element (CRE), oct-l, cjun and a metal regulatory element (MRE). The mtl-l gene is organized similarly; it is 440 bp and contains S'-CRE and oct-l but surprisingly, lacks an MRE. The mtl-2 promoter /enhancer was fused with the , Bgalactosidase (,B-gal) reporter gene and introduced into stable transgenic worms. High-level expression of ,B-gal was selectively induced in intestinal cells by Cd. This suggests that a single cell type might confer the initial line of defense against heavy metal toxicity. Three SIP cDNAs were isolated by differentially screening a cDNA library corresponding to mRNA templates from Cd-treated C. elegans. SIP-l encodes a novel low Mr 'stress protein' related to C. elegans heat shock proteins; SIP-2 encodes a putative secreted proteolytic aspartyl proteinase-like enzyme; SIP-3 encodes the catalytic (C) subunit of protein kinase A. The activity, mass and mRNA for C are increased 10-20-fold in Cd-adapted C. elegans. These results suggest that resistance to Cd toxicity in C. elegans is a complex process that entails the coordinated regulation of the expression of numerous genes. The atypical elevation in C and the CRE elements upstream from mtl-l and mtl-2 raises the possibility that the persistent activation of PKA is essential for a coordinated, adaptive transcriptional response to Cd.

Damijonaitis A et al. (2015) ACS Chem Neurosci "AzoCholine Enables Optical Control of Alpha 7 Nicotinic Acetylcholine Receptors in Neural Networks."

Broichhagen J, Urushima T, Nagpal J, Damijonaitis A, Laprell L, Sumser MP, Rafiq A, Gottschalk A, Trauner D, Hull K, Schonberger M, Kummer W, Woodmansee DH

[ACS Chem Neurosci, 2015]

Nicotinic acetylcholine receptors (nAChRs) are essential for cellular communication in higher organisms. Even though a vast pharmacological toolset to study cholinergic systems has been developed, control of endogenous neuronal nAChRs with high spatiotemporal precision has been lacking. To address this issue, we have generated photoswitchable nAChR agonists and re-evaluated the known photochromic ligand, BisQ. Using electrophysiology, we found that one of our new compounds, AzoCholine, is an excellent photoswitchable agonist for neuronal 7 nAChRs, whereas BisQ was confirmed to be an agonist for the muscle-type nAChR. AzoCholine could be used to modulate cholinergic activity in a brain slice and in dorsal root ganglion neurons. In addition, we demonstrate light-dependent perturbation of behavior in the nematode, Caenorhabditis elegans.

Reischl M et al. (2019) PLoS Comput Biol "Motion prediction enables simulated MR-imaging of freely moving model organisms."

Fuhrer E, Bartschat A, MacKinnon N, Mikut R, Jouda M, Reischl M, Korvink JG, Bakhtina N

[PLoS Comput Biol, 2019]

Magnetic resonance tomography typically applies the Fourier transform to k-space signals repeatedly acquired from a frequency encoded spatial region of interest, therefore requiring a stationary object during scanning. Any movement of the object results in phase errors in the recorded signal, leading to deformed images, phantoms, and artifacts, since the encoded information does not originate from the intended region of the object. However, if the type and magnitude of movement is known instantaneously, the scanner or the reconstruction algorithm could be adjusted to compensate for the movement, directly allowing high quality imaging with non-stationary objects. This would be an enormous boon to studies that tie cell metabolomics to spontaneous organism behaviour, eliminating the stress otherwise necessitated by restraining measures such as anesthesia or clamping. In the present theoretical study, we use a phantom of the animal model C. elegans to examine the feasibility to automatically predict its movement and position, and to evaluate the impact of movement prediction, within a sufficiently long time horizon, on image reconstruction. For this purpose, we use automated image processing to annotate body parts in freely moving C. elegans, and predict their path of movement. We further introduce an MRI simulation platform based on bright field videos of the moving worm, combined with a stack of high resolution transmission electron microscope (TEM) slice images as virtual high resolution phantoms. A phantom provides an indication of the spatial distribution of signal-generating nuclei on a particular imaging slice. We show that adjustment of the scanning to the predicted movements strongly reduces distortions in the resulting image, opening the door for implementation in a high-resolution NMR scanner.

Laura Herndon et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "What's New on WormAtlas and WormImage?"

Zeynep Altun, Tylon Stephney, Carolyn Norris, Laura Herndon, David Hall, Chris Crocker

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

We are updating the WormAtlas website to give it a fresh look. This is the first major revamping of WormAtlas since its launch in 2002. These changes will start from the front page and then be implemented throughout the Handbook and many other portions of the website. Here we will explain the principles of the new organization, and show you how to find your favorite features. We hope you will find the site simpler to navigate and we expect it will be more intuitive for beginners. Inside the WormAtlas website, there will be several major changes. First will be an improved adult hermaphrodite handbook; it will include several completely revised chapters and a new one covering the nervous system. Second will be the launch of a handbook for anatomy of the worm embryo. Third will be the addition of more data to our slice-by-slice worm viewer, Slidable Worm. Lastly, we will be adding many new Neuron pages for the male nervous system in order to highlight new synaptic patterns emerging from the Wired Worm project conducted together with Scott Emmons. The WormImage website, which houses thousands of unpublished electron micrographs and related data, is expanding steadily. It now presents much more data from mutant animals, particularly for genes affecting the nervous system. We continue to rely heavily on MRC datasets, but we are also adding more from the Riddle and Hall lab files, among others. We encourage more laboratories to share their own best archival TEM and SEM images so that this resource can continue to grow and serve the C. elegans community. We are very grateful to many labs that have already contributed ideas, advice and experimental results that are featured on these websites. This work is supported by NIH RR12596.

Lerchner W et al. (2007) Neuron "Reversible silencing of neuronal excitability in behaving mice by a genetically targeted, ivermectin-gated Cl- channel."

Anderson DJ, Slimko EM, Lerchner W, Lester HA, Xiao C, van Trigt L, Nashmi R

[Neuron, 2007]

Several genetic strategies for inhibiting neuronal function in mice have been described, but no system that directly suppresses membrane excitability and is triggered by a systemically administered drug, has been validated in awake behaving animals. We expressed unilaterally in mouse striatum a modified heteromeric ivermectin (IVM)-gated chloride channel from C. elegans (GluClalphabeta), systemically administered IVM, and then assessed amphetamine-induced rotational behavior. Rotation was observed as early as 4 hr after a single intraperitoneal IVM injection (10 mg/kg), reached maximal levels by 12 hr, and was almost fully reversed by 4 days. Multiple cycles of silencing and recovery could be performed in a single animal. In striatal slice preparations from GluClalphabeta-expressing animals, IVM rapidly suppressed spiking. The two-subunit GluCl/IVM system permits "intersectional" strategies designed to increase the cellular specificity of silencing in transgenic animals.

Bergs ACF et al. (2023) Nat Commun "All-optical closed-loop voltage clamp for precise control of muscles and neurons in live animals."

Dill H, Zeitzschel N, Vierock J, Liewald JF, Bessel A, Wirt C, Durmaz H, Bergs ACF, Nozownik A, Jospin M, Liu Q, Hegemann P, Bargmann CI, Rodriguez-Rozada S, Wiegert JS, Gottschalk A

[Nat Commun, 2023]

Excitable cells can be stimulated or inhibited by optogenetics. Since optogenetic actuation regimes are often static, neurons and circuits can quickly adapt, allowing perturbation, but not true control. Hence, we established an optogenetic voltage-clamp (OVC). The voltage-indicator QuasAr2 provides information for fast, closed-loop optical feedback to the bidirectional optogenetic actuator BiPOLES. Voltage-dependent fluorescence is held within tight margins, thus clamping the cell to distinct potentials. We established the OVC in muscles and neurons of Caenorhabditis elegans, and transferred it to rat hippocampal neurons in slice culture. Fluorescence signals were calibrated to electrically measured potentials, and wavelengths to currents, enabling to determine optical I/V-relationships. The OVC reports on homeostatically altered cellular physiology in mutants and on Ca²⁺-channel properties, and can dynamically clamp spiking in C. elegans. Combining non-invasive imaging with control capabilities of electrophysiology, the OVC facilitates high-throughput, contact-less electrophysiology in individual cells and paves the way for true optogenetic control in behaving animals.