Li, Zihao et al. (2021) International Worm Meeting "A robotic system for automated manipulation of C. elegans on agar media"

Bowlin, Peter, Li, Zihao, Fouad, Anthony, Du, Angelica, Kassouni, Alexander, Bhirgoo, Priya, Teng, Christopher, Fang-Yen, Christopher

[International Worm Meeting, 2021]

The time and labor required for worm picking is a major bottleneck for many C. elegans experiments, especially those requiring a large number of strains. Many genetic screens and genetic manipulations would benefit greatly from an automated method for worm pushing. We developed a robot capable of transferring worms between agar plates using movements similar to those used for manual worm picking. The robot contains a motorized 3D stage that positions a wire loop pick mounted on a robotic arm to manipulate worms on an array of standard plates. Capacitive touch sensing is used to monitor contact between the pick and the agar substrate and provide feedback for the fine movements needed for picking. We constructed a dual-magnification fluorescence and bright field microscope capable of identifying developmental, morphological, or fluorescence based phenotypes of individual worms at high resolution while simultaneously imaging the entire plate at low resolution. We developed software to identify, classify, and track worms using a combination of machine vision methods, including motion detection, adaptive thresholding, and a convolutional neural network trained to recognize worms. In a test of its fluorescence-based sorting capabilities, the robot accurately identified, phenotyped, picked, and transferred worms to other plates at a rate of about 3 animals per minute. We are developing a high-level scripting language that will enable the robot to autonomously perform multi-step procedures, such as integrating extrachromosal arrays, performing genetic crosses, generating clonal populations of mutagenized worms, and other tasks. Automation of worm manipulation will both increase researchers' productivity and enable experiments that are impractical using standard methods.

Hsieh, Yi-Wen et al. (2013) International Worm Meeting "tld-1 regulates the synaptic localization of the TIR-1 Ca2+ signaling scaffold protein in left-right neuronal asymmetry."

Chang, Chieh, Chuang, Chiou-Fen, Hsieh, Yi-Wen

[International Worm Meeting, 2013]

The C. elegans left and right AWC olfactory neurons specify asymmetric subtypes through a stochastic, coordinated cell signaling event. We previously showed that the TIR-1/Sarm1 scaffold protein assembles a Ca2+-regulated signaling complex at synaptic regions in the AWC axons, in a microtubule-dependent mechanism, to specify AWC diversity. We also showed that proper localization of the TIR-1 Ca2+ signaling complex at the AWC synapses is important for precise AWC asymmetry. To identify additional molecules required for the localization of TIR-1 in AWC subtype choice, we performed an unbiased forward genetic screen to identify mutants with defective TIR-1 localization and AWC diversity. From the screen, we identified tld-1, tld-2, tld-3, and tld-4 (TIR-1 localization defective) with reduced localization of TIR-1 at synapses in the AWC axons, and/or accumulation of TIR-1 in the AWC cell body. We have identified the mutation responsible for the tld-1 phenotype using whole genome sequencing (kindly performed by Alexander Boyanov in Oliver Hober's lab). We will present genetic analysis of tld-1 mutants and molecular characterization of the tld-1 gene in the specification of left-right AWC neuronal asymmetry. Our study of the tld genes will provide insights into how cell-specific Ca2+ signaling proteins such as TIR-1 are linked to the conserved synaptic assembly in neuronal diversification.

Kamat, Shaunak et al. (2013) International Worm Meeting "Modulation of mec-10(d)-induced necrosis by ER chaperone NRA-2."

Driscoll, Monica, Yeola, Shrutika, Bianchi, Laura, Kamat, Shaunak

[International Worm Meeting, 2013]

ER chaperones play a major role in cellular homeostasis by regulating the assembly of polypeptides, intracellular Ca2+ signaling, and degradation of misfolded proteins. Here we report on an ER - resident chaperone NRA-2 that modulates hyper-activated ion channel-induced necrosis by regulating surface expression of a death-inducing DEG/ENaC channel family member subunit MEC-10(d). nra-2 was previously identified in a screen for nicotinic acetylcholine receptor (nAChR) - interacting proteins and was found to regulate the subunit composition of the AChR receptor in C. elegans muscle1. We found that loss of function of nra-2 led to a significant increase in mec-10(d) - induced necrosis in C. elegans touch receptor neurons (TRNs), and this enhancement was rescued by TRN-specific transgenic expression of nra-2. We observed that loss of nra-2 led to a significant increase in surface localization of MEC-10(d)::GFP and a significant decrease in ER localization. Electrophysiological experiments in Xenopus oocytes revealed that NRA-2 suppresses amiloride-sensitive currents induced by hyperactivated MEC channels. Our study suggests a role for NRA-2 as an ER quality control protein that inhibits surface expression of mutant MEC-10(d) channels and is thus neuroprotective against hyperactivated ion channel-induced necrosis. 1. Ruta B. Almedom, Jana F. Liewald, Guillermina Hernando, Christian Schultheis, Diego Rayes, Jie Pan, Thorsten Schedletzky, Harald Hutter, Cecilia Bouzat, and Alexander Gottschalk. An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse. The EMBO Journal, 28(17):2636-2649, July 2009.

Stefan Eimer et al. (2006) European Worm Meeting "The Integral Membrane Protein UNC-50 is required for Nicotinic Receptor Trafficking in C. elegans"

Janet Richmond, Alexander Gottschalk, Michael Hengartner, Stefan Eimer, Jean-Louis BESSEREAU, William R Schafer

[European Worm Meeting, 2006]

Stefan Eimer1, Alexander Gottschalk2, Janet Richmond3, Michael Hengartner4, William R Schafer2, and Jean-Louis Bessereau1. Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that undergo multiple assembly, modification and transport steps prior to insertion into the plasma membrane. However, little is known about the molecules that are specifically participating in these processes. In C. elegans a nAChR sensitive to the nematode-specific cholinergic agonist levamisole has been well characterised. Mutants in all of its four subunits confer resistance to muscle hyper-contraction and death upon exposure to levamisole.. Similarly, unc-50 mutant animals exhibit the same resistance to paralysis and death upon exposure to levamisole. By antibody staining and electro-physiological recording of body-wall muscles we show that levamisole receptors are not detectable at cell surface in unc-50 mutants. However, a second type of muscle nAChR, which is insensitive to levamisole, and the GABA receptor are normally transported to the synapse. unc50 encodes an integral membrane protein conserved from yeast to man. In C. elegans, unc-50 is ubiquitously expressed and localized to the Golgi system. We showed that UNC-50 is required in body wall muscles for the transport of the assembled levamisole receptor to neuromuscular junction. In the absence of UNC-50 the receptor is sorted within Golgi to the lysosomal system and rapidly degraded. Therefore, UNC-50 might be part of/constitute a Golgi resident check point during levamisole receptor transport. UNC-50 interacts with the GBF1 class of large Arf GEFs which are involved in retrograde transport within the Golgi system. Models how retrograde transport and sorting would required for levamisole receptor trafficking will be discussed.

Chan, Kevin K et al. (2011) International Worm Meeting "Integrin Cooperates with UNC-40 to Guide Muscle Arm Extension."

Roy, Peter J, Chan, Kevin K

[International Worm Meeting, 2011]

Body muscles of nematodes extend specialized plasma membranes, called muscle arms, to the dorsal and ventral nerve cords to form neuromuscular junctions. The extension of C. elegans muscle arms is stereotypical and likely guided by a chemoattractant that is secreted from motor axon targets. We recently found that an UNC-40/DCC pathway, which includes the UNC-40 receptor, the MADD-2 adaptor protein, and the UNC-73 Rho-GEF, functions cell-autonomously in C. elegans to direct muscle arm extension towards the motor axons (Alexander et al., 2009, 2010). Two lines of evidence suggest that this signalling pathway may be a non-canonical one. First, UNC-6/Netrin, which is the canonical ligand of UNC-40, is dispensable for muscle arm extension to motor axons. Second, the extra-cellular domains of UNC-40 are not required for UNC-40's role in directing muscle arm extension. These data suggest that a second receptor complex may co-operate with UNC-40 to guide muscle arms. To identify other plasma membrane-associated proteins that might cooperate with UNC-40, we screened candidate receptors for their ability to facilitate UNC-40-mediated muscle arm extension. We previously reported that PAT-2/a-integrin, but not INA-1/a-integrin, is required for muscle arm extension to the motor axons (Dixon et al., 2006). Here, we present evidence that the PAT-2/PAT-3 integrin heterodimer is required for UNC-40-dependent muscle arm extension. We are currently investigating the mechanism by which integrin facilitates UNC-40's role in directing muscle arm extension.

Martin Brauner et al. (2006) European Worm Meeting "Exogenous Stimulation of Muscles and Neurons via Photoactivation of Channelrhodopsin-2 Triggers Rapid Behavioural Responses"

Martin Brauner, Jana Liewald, Alexander Gottschalk

[European Worm Meeting, 2006]

Martin Brauner, Jana Liewald and Alexander Gottschalk. Except for sensory neurons responding to specific stimuli, there are no satisfying methods available to precisely stimulate certain neurons in C. elegans, neither in vivo, nor in dissected animals. We developed a method that allows to stimulate distinct neurons (or other excitable cells, i.e. muscle cells) in C. elegans, and thus to trigger specific behaviours, simply by illuminating the animals with blue light. To this end, we heterologously expressed the protein Channelrhodopsin-2 (ChR2), a light-gated cation channel from the green alga Chlamydomonas reinhardtii, using cell-(type)specific promoters. Illumination of animals expressing ChR2 in muscles led to immediate muscle contractions and thus to a readily observable shrinking of the whole body, when animals were cultured in the presence of the chromophore all-trans retinal (required for ChR2 function). Electrophysiological whole-cell recordings from body-wall muscle cells showed rapid inward currents persisting as long as the illumination. Animals expressing ChR2 in mechanosensory neurons showed withdrawal behaviours after illumination, that were reminiscent of the response to mechanical stimulation (gentle touch, i.e. or more precisely, tapping of the culture dish). These behaviours could also be triggered in mutants lacking functional MEC-4/MEC-10 mechanosensory ion channels, indicating that the touch receptor neurons were still functionally integrated in their circuit. Besides sensory neurons, preferential expression and photoactivation of ChR2 in the (backward) command interneurons AVA and AVD (using the nmr-1 promoter), also led to rapid reversal behaviour. We are further expressing ChR2 in cholinergic motorneurons and expect to thus trigger acetylcholine release and, consequently, muscle contractions. In sum, the heterologous expression of ChR2 in C. elegans enables the specific stimulation of excitable cells by light, which should allow to trigger any neuron (provided a specific promoter is available) and to study the (behavioural) output of this cell, potentially also by imaging of neuronal activity in downstream cells using the genetically encoded optical Ca2+-sensor Cameleon.

Panagiotis Psarrakos et al. (2006) European Worm Meeting "Disulphide Bond Formation in Multicellular Organisms"

Panagiotis Psarrakos, Dr. Peter Klappa.

[European Worm Meeting, 2006]

Proteins are vitally important macromolecules that perform diverse roles in all organisms. A common feature of any protein is its requirement for a specific three-dimensional structure for biological function. In many secreted and membrane-associated proteins this three-dimensional structure is stabilised by intra- or intermolecular disulphide bonds, which are usually formed post-translationally in the endoplasmic reticulum (ER). Research currently indicates that disulphide bond formation can occur via multiple parallel pathways where a large number of protein families may play a role in supplying redox equivalents. Furthermore, late-stage isomerization reactions, where disulphide bond formation is commonly linked to conformational changes in protein substrates that possess significant secondary structures, are thought to be catalysed by members of the protein disulphide isomerase (PDI) family.. Several proteins with similarity to PDI have been described in the ER of higher eukaryotes. The precise in vivo functions of these proteins remain elusive and the question arises why there are different members of the PDI family present in the same intracellular compartment. One inevitable speculation is that they have overlapping biological functions with their distinct roles only coming into effect under specific conditions, but this has not to date been systematically examined. Caenorhabditis elegans contains a full set of proteins with high homology to the mammalian members of the PDI family and to members of other protein families actively involved in disulphide redox reactions and thus, is invaluable as a eukaryotic model system for their biological and biochemical characterisation.. We report here on the RNAi analysis of members of the PDI family and of other proteins involved in disulphide bond formation in a multicellular organism. In addition, biochemical characterization of the major members of the PDI family in C. elegans, coupled with analysis of loss-of-function phenotypes (either RNAi-induced or based on genomic knockouts), strongly indicates an overlap in substrate specificities and, potentially, functions.. I would like to thank the Alexander S. Onassis Public Benefit Foundation for awarding me with a scholarship for the duration of this project.

Thorsten Schedletzky et al. (2006) European Worm Meeting "Characterization of SOC-1 and PLK-2, Two Proteins Modulating Levamisole-Receptor Function in C. elegans"

Alexander Gottschalk, Thorsten Schedletzky

[European Worm Meeting, 2006]

Thorsten Schedletzky and Alexander Gottschalk. We have previously identified novel proteins associated with the levamisole receptor, one of two nicotinic acetylcholine receptors (nAChRs) acting at the neuromuscular junction (NMJ) of Caenorhabditis elegans, using a tandem affinity approach, mass spectromety and RNAi-based functional screening (Gottschalk et al., 2005). Two of the proteins that show altered nicotine-sensitivity after knock-down are the multisubstrate adaptor protein SOC-1 and the polo-like kinase PLK-2. soc-1(n1789) mutants show resistance to nicotine and levamisole in paralysis assays. As it is well known that SOC-1 acts downstream of the EGL-15 fibroblast growth factor (FGF) receptor tyrosine kinase (RTK), we tested egl-15 as well as other positive regulators of the FGF pathway. We could show that soc-1 is connected not only to EGL-15 signalling in clustering of levamisole receptors at the NMJ, but also to a second RTK, CAM-1, and that it also affects clustering of GABAA receptors, potentially through a third, as yet unknown RTK. A SOC-1::GFP fusion is strongly expressed in amphid sheath and socket cells, but nevertheless rescues the soc-1 phenotype in clr-1; soc-1 double mutants. We try to detect SOC-1 in other cells, using a specific antibody.. Currently, we are screening for other receptor tyrosine kinases in C. elegans causing a nicotine or levamisole resistance phenotype, to identify novel upstream regulators of SOC-1, that may be involved in nAChR and/or GABAA receptor clustering at the NMJ.. PLKs are kinases generally involved in cell-division and chromosome segregation, however, vertebrate PLK-2 homologues (Fnk and Snk) also affect synaptic plasticity. plk-2(tm1395) mutants are weakly nicotine and levamisole resistant. Characterization of plk-2(tm1395) by electrophysiology, however, indicated only a trend towards reduced currents induced by cholinergic agonists in muscle (see abstract by J. Liewald). plk-2 mutants also show a high incidence of males (him) phenotype, indicating indeed a role in centrosome duplication and chromosome segregation. Consistently, PLK-2::GFP appears to be expressed both in dividing and non-dividing cells, including muscle.. Reference: Gottschalk A, Almedom R, Schedletzky T, Anderson SD, Yates JR, and Schafer WR (2005) EMBO J 24: 2566-2578

Ji Li et al. (2005) International Worm Meeting "ftt-2 plays dual roles in daf-2/insulin-like signaling pathway"

Ji Li, Siu Sylvia Lee, Muneesh Tewari, Marc Vidal

[International Worm Meeting, 2005]

To investigate additional components that may regulate the daf-2/insulin-like signaling pathway, we performed a small scale RNAi screen focusing on putative signal transduction molecules. We found that RNAi inactivation of ftt-2 enhanced the dauer formation phenotype of the daf-2(e1370) temperature sensitive mutant at the semi-restrictive temperature. In addition, RNAi knock down of ftt-2 stimulated DAF-16::GFP nuclear accumulation. ftt-2 encodes a 14-3-3 protein. The 14-3-3 family of proteins are structurally and functionally highly conserved. In contrast to ftt-2, RNAi inactivation of par-5/ftt-1, the only other 14-3-3 protein in C. elegans, did not show notable effects on dauer formation phenotype or DAF-16::GFP, suggesting that FTT-2 specifically regulates the daf-2/insulin-like signaling pathway. Mammalian 14-3-3 has previously been shown to bind to DAF-16 in vitro (1), and in mammalian cells, 14-3-3 sequesters the DAF-16 orthologs FOXOs in the cytoplasm (2). Therefore, our results suggest that in C. elegans, FTT-2 also regulates daf-2/insulin-like signaling by sequestering the forkhead transcription factor DAF-16 from the nucleus. Interestingly, RNAi knock down of ftt-2 also resulted in dramatic decrease of DAF-16 expression levels. We demonstrated that ftt-2 RNAi specifically affected DAF-16 protein levels, but not daf-16 RNA levels. Considering the recent findings in mammalian cells indicating that the FOXOs proteins are targeted for polyubiquitin-mediated protein degradation, we propose that, in C. elegans, the regulation of DAF-16 levels by FTT-2 is likely through ubiquitin/proteasome degradation. Therefore, in C. elegans, FTT-2 is able to regulate DAF-16 via cytoplasmic sequestration, as well as protection from protein degradation. References: 1) Catherine M. Cahill, Guri Tzivion, Nargis Nasrin, Scott Ogg, Justin Dore, Gary Ruvkun, and Maria Alexander-Bridges. Phosphatidylinositol 3-Kinase Signaling Inhibits DAF-16 DNA Binding and Function via 14-3-3-dependent and 14-3-3-independent Pathways. J. Biol. Chem., Vol. 276, 13402-13410, 2001 2) Anne Brunet, Fumihiko Kanai, Justine Stehn, Jian Xu, Dilara Sarbassova, John V. Frangioni, Sorab N. Dalal, James A. DeCaprio, Michael E. Greenberg and Michael B. Yaffe. 14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport. The Journal of Cell Biology, Vol. 156, 817-828, 2002

Jana Liewald et al. (2006) European Worm Meeting "Electrophysiological Analysis of C. elegans Mutants in Levamisole-Receptor Associated Proteins"

Ruta Almedom, Thorsten Schedletzky, Jana Liewald, Alexander Gottschalk

[European Worm Meeting, 2006]

Jana Liewald, Ruta Almedom, Thorsten Schedletzky and Alexander Gottschalk. We have previously identified proteins associated with the levamisole receptor, one of two nicotinic acetylcholine receptors (nAChR) acting at the neuromuscular junction of Caenorhabditis elegans, after tandem affinity purification, mass spectrometry, and subsequent RNAi-based functional screening (Gottschalk et al., 2005). For proteins causing altered nicotine-sensitivity in vivo, genomic (deletion) mutants were obtained. Currently, we are analyzing these mutants electrophysiologically for the effects on the functional properties of nAChRs (and GABAA receptors) present at the neuromuscular junction. Therefore, body wall muscle cells of C. elegans are whole cell patch-clamped and inward currents are determined, induced by application of neurotransmitters (ACh, GABA) and other cholinergic agonists (levamisole, nicotine).. Preliminary data indicate that the nra-2(tm1453) mutant shows significantly reduced currents mediated by ACh and levamisole, while GABA-induced currents are like in wild-type animals. This is in agreement with other functional assays for sensitivity to such agonists carried out in our group: nra-2(tm1453) mutants show moderate resistance to paralysis induced by levamisole and nicotine, as well as significantly reduced levamisole receptor levels at the neuromuscular junction (found after in vivo fluorescence labeling). Sensitivity to muscimol, a GABAA receptor agonist, however, was not affected in nra-2(tm1453) animals.. Loss of the POLO-like kinase PLK-2, which had caused weak nicotine resistance in our paralysis assays (see also abstract by T. Schedletzky), did not show significantly reduced inward currents in body wall muscle cells after application of cholinergic agonists, though a trend towards reduced currents became apparent.. Other mutants studied are a membrane-binding Copine (NRA-1) and the receptor-tyrosine kinase signalling protein SOC-1. Loss of either protein causes reduced synaptic levamisole receptor levels, while soc-1 mutants are also affected for synaptic GABAA receptor expression. Their electrophysiological evaluation is in progress and results will be presented at the meeting. References: Gottschalk, A., Almedom, R. B., Schedletzky, T., Anderson, S. D., Yates III, J. R., and Schafer, W. R. Identification and characterization of novel nicotinic receptor-associated proteins in Caenorhabditis elegans. EMBO J. 24 (2005), 2566-2578.