Mitchell DH (1977) International C. elegans Meeting "a nematode variant with increased lifespan."

Mitchell DH

[International C. elegans Meeting, 1977]

Mitchell, J. et al. (2019) International Worm Meeting "The role of cadherins at sensory synapses."

Teschner, L., Agenor, V., Benedetti, K., Bi, V., VanHoven, M., Tang, A., Park, J., Andersen, K., Muqtadir, T., Ali, N, Mitchell, J.

[International Worm Meeting, 2019]

The nervous system mediates such fundamental processes as sensory perception. The faithful formation of neural circuits is required for all forms of sensory perception, and defects in the formation and structure of neural circuits are thought to underlie neurological disorders, such as schizophrenia and autism spectrum disorder. Cadherins are a large superfamily of transmembrane cell adhesion molecules widely known for the roles they play during development. Cadherins are also known for their role in providing structural support, and have been implicated in synapse formation and integrity. However, much remains to be learned about the roles of individual cadherins in the formation of sensory circuits. We sought to identify cadherins necessary for the formation and integrity of sensory synapses using the phasmid sensory circuit in C. elegans. Specifically, we focused on synaptic connections between PHB sensory neurons, and AVA interneurons. To determine if defects in cadherins affect sensory synapses, we utilized the split GFP-based trans-synaptic marker NLG-1 GRASP (Neuroligin-1 GFP Reconstitution Across Synaptic Partners). We screened mutants affecting C. elegans cadherin superfamily proteins to identify molecules with altered NLG-1 GRASP intensity. The mutants we tested include the Dachsous-like cadherin cdh-1; the Fat-like cadherins cdh-3 and cdh-4; the nematode-specific cadherins cdh-5, cdh-7, cdh-8, and cdh-10; the Flamingo/Stan cadherin cdh-6, and cdh-9, which is similar to DCad96Cb. We found modest decreases in NLG-1 GRASP intensity in the nematode-specific cadherins cdh-5 and cdh-8, and an approximately 50% decrease in NLG-1 GRASP intensity in cdh-6/Flamingo, which was previously shown to regulate synapse development in VD motorneurons by Najarro and colleagues (J. Neurosci, 2012). Interestingly, defects in cdh-6/Flamingo were present in L4, but not L1 animals, suggesting a later function for this gene at PHB-AVA synapses. Identifying roles for cadherins at sensory synapses will bring us closer to understanding the factors that may be playing roles in synaptic maintenance across an organism's lifetime, and may also help us better understand mechanisms whose dysfunction leads to neurological disorders.

Romens, Mitchell A. et al. (2011) International Worm Meeting "mir-237 is regulated by lin-14 and functions in early developmental timing."

Romens, Mitchell A., Abbott, Allison L., Kemp, Benedict J.

[International Worm Meeting, 2011]

In C. elegans, appropriate control of two key developmental timing regulators, lin-14 and lin-28, involves the activities of the lin-4 microRNA and a lin-4 independent positive feedback loop between lin-14 and lin-28. This lin-4 independent feedback loop is likely mediated by microRNA activity. However, the regulators of the positive feedback loop have not been identified. We are testing the hypothesis that the lin-4 family member, mir-237, functions in the positive feedback loop. We found that loss of mir-237 enhances the lin-46 retarded extra seam cell phenotype, indicating a reiteration of the L2 stage program. This enhancement is dependent on lin-28 activity. In addition, we found that loss of mir-237 activity in worms lacking lin-4 and with reduced activity of lin-14 results in extra seam cells. We also showed that loss of mir-237 is able to partially suppress the precocious phenotype of a lin-14 reduced function allele, indicating that mir-237 may function downstream of lin-14. Interestingly, mir-237 expression is greatly reduced in lin-4 mutant worms (Esquela-Kerscher et al., Developmental Dynamics 234:868-877, 2005) in which LIN-14 and LIN-28 protein levels are elevated. Using qRT-PCR we found that in lin-14 null worms, mir-237 expression is increased more than 4-fold relative to wild-type controls at the L1 stage. We also show that in lin-14 gain-of-function worms, mir-237 levels are substantially reduced relative to wild-type. Similar results are observed using a mir-237 transcriptional reporter, indicating that mir-237 transcription is repressed by lin-14 activity. Our current model is that LIN-14 acts, directly or indirectly, to repress mir-237 expression during the L1 stage and as LIN-14 levels drop in the L2 stage, mir-237 begins to be expressed and can then act to repress lin-28 to control the L2-to-L3 transition.

Mitchell PH et al. (2008) European Worm Meeting "Modelling alcohol dependence in Caenorhabditis elegans"

James C, Mitchell PH, Mould RN, Hopper NA, Glautier SP, Dillon J, Oconnor VM, Holden-Dye LM,, Adrianakis I

[European Worm Meeting, 2008]

Ethanol is one of the most widely used and socially acceptable drugs in the. world. However its chronic use can lead to serious problems due to the. development of dependence. Ethanol has been shown have wide-ranging but. selective effects on many different neurotransmitter circuits including;. glutamatergic, GABAergic, serotonergic, dopaminergic and opioid peptidergic. systems. The basis for alcohol tolerance and withdrawal involves. counteradaptive neurochemical changes within these systems to adapt to the. prolonged presence of ethanol in the brain. Here we are using C. elegans to. enable an analysis at all levels of organization from gene, molecule, and. neurone through to neuronal circuit and behaviour. We want to use C.. elegans to investigate the development of neuroadaptation to ethanol. In. order to achieve this we have first provided evidence that ethanol rapidly. equilibriates across the cuticle of C. elegans (Mitchell et al. 2007) thus. enabling experiments in which the concentration-dependent effects of. ethanol on behaviour can be defined. In line with previous studies (Davies. et al. 2003) we have established that external concentrations of ethanol. >100mM are required to overtly affect the visually observable measures of. behaviour. This is equivalent to concentrations that are supra-intoxicating. in mammals. In order to determine whether ethanol elicits any effects on. C. elegans at concentrations equivalent to those that are intoxicating in. mammals we have performed more discreet analyses of behaviours. Pharyngeal. recordings have revealed that concentrations as low as 10mM ethanol have. significant effects on the activity of neural networks, thus providing an. opportunity to investigate the molecular determinants of acute ethanol. effects. We are also in the process of refining the analysis of locomotory. behaviour with the aim of determining whether ethanol elicits effects at. low mM concentrations. As part of this effort we have developed a paradigm. in which we can demonstrate withdrawal from ethanol, and relief from. withdrawal due to low concentrations of acute ethanol. We have used this to. study the onset and recovery rates of this neuroadaptation and the. concentration dependence of this effect in order to understand the. mechanisms involved. This has been extended to investigate if genes already. implicated in acute effects of ethanol in C. elegans are involved in. controlling the withdrawal and tolerance behaviours. These studies are. aimed at better defining how well C. elegans can model aspects of. alcoholism.. Funded by the BBSRC, UK.. References. Davies, A. G., Pierce-Shimomura, J. T., Kim, H., VanHoven, M. K., Thiele,. T. R., Bonci, A., Bargmann, C. I., & McIntire, S. L. 2003, "A central role. of the BK potassium channel in behavioral responses to ethanol in C.. elegans", Cell, vol. 115, no. 6, pp. 655-666.. Mitchell, P. H., Bull, K., Glautier, S., Hopper, N. A., Holden-Dye, L., &. O''connor, V. 2007, "The concentration-dependent effects of ethanol on. Caenorhabditis elegans behaviour", Pharmacogenomics.J., vol. 7, no. 6, pp.. 411-417.

Mitchell, Robin et al. (2021) International Worm Meeting "Reversal behavior upon encountering a cliff involves mechanosensation"

Mitchell, Robin, Young, Jared, Zhang, Shuyu, Pattillos, Diana

[International Worm Meeting, 2021]

The precipice response is a little-understood phenomenon in which C. elegans move rapidly away from edges or gaps in an agar plate. First described by Chalfie et al. (2014), this behavior has yet to be characterized by the research community, and up until now potential underlying mechanisms remained unexplored. We hypothesized that mechanosensation underlies the precipice response and that mutant worms with deficient mechanosensation would exhibit the precipice response less frequently than N2 wild-type worms. Our experiments sought to 1) characterize the precipice response and craft a sufficient biological definition, and 2) test our hypothesis about mechanosensation's role in the precipice response. N2 wild-type worms were used to define the precipice response. Using a wire or dog whisker pick, worms were transferred onto small chunks of agar with smooth, 90° edges. We observed that, indeed, worms had a strong tendency to quickly reverse when their head went over the edge of the agar. We defined the precipice response as a reversal initiated within two seconds of the head going over a 90° edge. This reversal must extend for a full sine wave within two seconds. In an initial dataset of 32 N2 worms, 26 of them (81%) exhibited the precipice response. To test whether mechanosensation plays an important role in the precipice response, we tested three strains of worms with varying degrees of mechanosensory deficiency: mec-3(e1338), mec-10(e1515), and trp-4(sy595). mec-3 mutants do not respond to light or harsh touch, while mec-10 and trp-4 mutants exhibit partial loss of response to mechanical stimuli. In blinded trials, we found that mec-3 mutants exhibited the precipice response less frequently than N2, mec-10, or trp-4 worms. Neither mec-10 nor trp-4 exhibited the precipice response at a frequency that was significantly different from the N2 strain. These results show that mechanosensation underlies the precipice response, and imply that near total loss of mechanosensation may be necessary to have a profound impact on the precipice response. This study is an illuminating first step in discerning the mechanisms of the precipice response in C. elegans, and may have broader implications in animal behavior.

Mitchell, Shyrice et al. (2017) International Worm Meeting "Bypass Suppressors of mrp-5: Identifying Novel Components of Heme Homeostasis."

Smith, Harold, Hamza, Iqbal, Krause, Michael, Mitchell, Shyrice

[International Worm Meeting, 2017]

Heme is a ubiquitously expressed biomolecule conserved in all eukaryotic systems. This cofactor is vital due to its essentiality in hemoproteins such as cytochromes, hemoglobin, and myoglobin. While it can be intuitively understood that heme is necessary for life because of the role hemoproteins play in the production of ATP, free heme is cytotoxic. Thus, cells must have the capability to transport, traffick, and chaperone heme. C. elegans are an excellent animal model to elucidate heme trafficking pathways because this roundworm is unable to synthesize its own heme but instead acquires it from the environment. By exploiting this heme auxotrophy, we built the first molecular framework for heme trafficking in eukaryotes. C. elegans acquires dietary heme via HRG-1-related transmembrane heme permeases into the worm intestine while heme is exported from the intestine to extraintestinal tissues by ABCC5/MRP-5. Loss of MRP-5 results in an embryonic-lethal phenotype because heme is trapped within the intestines but extraintestinal cells are heme deprived, a phenotype that can be suppressed by large amounts of heme supplementation. To uncover additional components of heme transport by MRP-5, a forward genetic screen was performed on mrp-5 (ok2067) deletion mutants to identify bypass suppressors of mrp-5-dependent lethality. Screening of 160,000 haploid genomes yielded thirty-two mrp-5(ok2067) suppressors and deep-sequencing variant analyses revealed that three of the suppressors were subunits of adapter protein complex 3 proteins involved in vesicular cargo sorting. We now propose to characterize the remaining suppressor mutants and identify their genetic lesions to construct a detailed cell biological pathway for organismal heme export.

P. Mitchell et al. (2006) European Worm Meeting "Evidence that Ethanol Equilibriates Rapidly Across C. elegans Cuticle"

L. Holden-Dye, N.A. Hopper, K. Bull, V. O'Connor, P. Mitchell, S. Glautier

[European Worm Meeting, 2006]

P. Mitchell, K. Bull, N.A. Hopper, S. Glautier1, L. Holden-Dye, V. O''Connor Ethanol has concentration-dependent effects on neural function. At concentrations in the low mM range, equivalent to the drink-driving limit, it has an intoxicating action, which is often associated with a transient excitability, followed by inhibition. At higher concentrations, greater than 100 mM, it has an anesthetic action and elicits muscle paralysis. C. elegans has been used to define the genetic basis for behavioural responses to ethanol exposure. In these studies intact animals have been exposed to 500 mM ethanol with the prediction that the internal concentration is more than ten-fold lower and therefore equivalent to an intoxicating, rather than an anesthetic, dose (Davies and McIntire, 2004). In order to underpin further studies, we have analysed the relationship between the external and internal ethanol concentration of C. elegans. Immersion of C. elegans in a sealed chamber containing ethanol (range 100 mM to 500 mM) elicits an inhibition of thrashing rate. Inhibition reaches a steady-state value within 5 min. This effect is concentration-dependent (half-maximal effect at 300 mM). At all concentrations of ethanol a steady-state inhibition is observed within 5 min. The animals rapidly recovered when removed from ethanol. Notably, this recovery was complete within 2.5 min. This suggests that ethanol may rapidly equilibriate across the cuticle and that the internal concentration that is required to inhibit motility approximates to the external concentration. In order to directly assay the effects of ethanol on muscle we used the pharyngeal muscle preparation. In this assay the muscle is exposed and therefore the concentration of ethanol is known. This muscle is inhibited by ethanol at concentrations in excess of 100 mM. Although it could be argued that the pharyngeal muscle behaves in a different fashion to body wall muscle in terms of ethanol sensitivity, the results support the contention that concentrations in excess of 100 mM are required to inhibit muscle activity. Internal ethanol concentrations in C. elegans following exposure to ethanol have been estimated using a biochemical assay kit. However, this procedure requires that the animals are briefly washed in cold buffer prior to the measurement. Our observation that animals exposed to ethanol fully recover from the inhibition of motility within 2.5 min suggests that a significant amount of ethanol may be lost from the inside of the animals during the protocol. We tested this by determining the influence of the wash volume on the apparent estimate in internal ethanol concentration. This shows that the estimate of internal ethanol concentration increases as the volume of the wash buffer decreases. This suggests that the measurement of internal ethanol concentration using this method underestimates the true internal concentration.. Davies, A.G. and McIntire, S.L. 2004, Biol. Proced. Online 6, 113-119.. Funded by the BBSRC, UK

Mitchell, Diana et al. (2013) International Worm Meeting "Methods to Study Toxic Transgenes: Analysis of Protease-Dead Separase in Membrane Trafficking."

Uehlein, Lindsey, Mitchell, Diana, Bembenek, Joshua

[International Worm Meeting, 2013]

The C. elegans embryo provides an ideal system to study the processes of egg activation and cell division. We have demonstrated a novel role for separase, a conserved protease that facilitates chromosome segregation, in the regulation of membrane trafficking during cortical granule exocytosis and cytokinesis. We are interested in determining the role of separase's protease function in the regulation of membrane trafficking. To address this, we generated protease-dead separase mutant tagged with GFP (GFP::SEP-1-PD). We find that GFP::SEP-1-PD accumulates more strongly than wt GFP::SEP-1 at the furrow and midbody during anaphase and cytokinesis, suggesting it could be substrate trapping and have a role in membrane trafficking. The expression of GFP::SEP-1-PD causes embryonic lethality, suggesting that it is a dominant-negative transgene that interferes with endogenous separase function. To enable further study of GFP::SEP-1-PD, we developed methods that allow propagation of worm lines containing toxic transgenes. We are using these methods to enable the study of the protease function of separase in membrane trafficking in the C. elegans embryo. The first method involves feeding worms gfp(RNAi) bacteria to silence the transgene. Transgene expression takes on average 5 generations after removal from gfp(RNAi). Transgene re-expression is also temperature sensitive, allowing further control over its re-expression. We are using this method to obtain large numbers of embryos expressing GFP::SEP-1-PD for biochemical and proteomics analysis, which may allow us to identify novel separase substrates. The second method takes advantage of the pie-1 promotor, which drives transgene expression, and is not expressed in the male germline. Male worms containing GFP::SEP-1-PD can used to propagate the transgene and circumvent the generation delay in the gfp(RNAi) feeding method. We are employing this method to examine genetic interactions of PD-separase with genes in the membrane trafficking pathway known to be involved in cortical granule exocytosis and/or cytokinesis. The general application of these methods facilitates the study of dominant-negative/toxic transgenes using standard techniques in the C. elegans model system.

Mitchell, Jacinth et al. (2011) International Worm Meeting "NRDE-1/MRT-4 defects result in a heritable toxic stress that can remodel germ cell development."

Godwin, Malik, Burkhart, Kirk, Hedges, Ashley, Liu, Yan, Ahmed, Shawn, Mitchell, Jacinth, Nakashima, Aisa, Simmons, Alicia, Kennedy, Scott

[International Worm Meeting, 2011]

Immortal germ cell lineages contain the capacity for unlimited proliferative ability, through successive generations. To better understand how proliferative aging is prevented in the germ line, mortal germline mutants were isolated, which show progressive sterility when propagated for multiple generations. Two alleles of mortal germline-4 (mrt-4), yp4 and yp5, were identified, which initially display normal levels of fertility and become sterile after growth for 8 to 14 generations at 20oC. As sterile mrt-4 mutants develop, L4 larvae with apparently normal germ lines mature into adults with few to no germ cells. To investigate the mechanism of this germline remodeling, double mutants of mrt-4 with ced-3, a caspase that is essential for apotosis, were constructed. Although alteration to the apoptotic cell death pathway does not suppress the progressive sterility phenotype of mrt-4, ced-3 abolished the germline restructuring phenotype observed in most sterile mrt-4 adults. Our results suggest that a heritable form of endogenous stress builds up in the absence of mrt-4, ultimately resulting in a caspase-mediated remodeling of the germline, which may correspond to a form of Adult Reproductive Diapause (1). mrt-4 was cloned and corresponds to nrde-1, a gene that is required for nuclear RNA interference (2). Although not all nrde genes are required for germ cell immortality, nuclear silencing defects may underlie the heritable form of toxic stress that causes sterility in mrt-4/nrde-1 strains.

Oakes, Mitchell et al. (2015) International Worm Meeting "Uncovering cannabinoid signaling in C. elegans, a new model to study the effects of medical Cannabis sativa."

Komuniecki, Richard, Law, Wen Jing, Ortega, Amanda, Oakes, Mitchell

[International Worm Meeting, 2015]

For millennia, marijuana, or Cannabis sativa, is used as a recreational drug, causing euphoria and altered sensory perception. C. sativa is also used for its unique medicinal effects and pure extracts of the plant have been used to treat conditions such as glaucoma, epilepsy, and neuropathic pain. Two primary bioactive compounds in C. sativa are delta9-tetrahydrocannabinol (THC) and cannabidiol (CBD), which significantly contribute to the plant's euphoric and medicinal effects, respectively. While the mechanisms by which THC and CBD exert their wide range of effects are not well understood, some of their molecular targets have been identified, including cannabinoid receptors 1 and 2 (CB1 and CB2). However, it is likely that additional, not yet identified, targets for both THC and CBD exist. In addition to exogenous agonists, CB1 and CB2 also bind several endogenous cannabinoids or endocannabinoids. In mammals, the major endocannabinoids 2-arachidonylglycerol (2-AG) and anandamide (AEA) activate the same signaling pathways as THC and CBD. The application of either 2-AG or AEA to wild-type C. elegans alters a variety of locomotory parameters. For example, both compounds inhibit locomotion and significantly increase the time taken to initiate a reversal to the noxious stimulus, 1-octanol. In addition, 2-AG also inhibits the serotonin stimulation of aversive responses to 1-octanol. The pre-synaptic enzyme monoacylglycerol lipase (MAGL) is responsible for degrading 2-AG and termination of endocannabinoid signaling. As expected, application of JZL184, a selective and irreversible inhibitor of MAGL, phenocopies the application of exogenous 2-AG. Preliminary studies have identified four C. elegans G-protein coupled receptors that are essential for various 2-AG and AEA dependent phenotypes. These studies are continuing with the in vitro characterization of these receptors as potential 2-AG and AEA receptors. As the number of states that legalize the medicinal use of C. sativa increases, so does the need for a better understanding of the receptors and signaling pathways mediating the effects of THC and CBD.