Ronan, E.A. et al. (2019) International Worm Meeting "Neural and genetic mechanisms of cinnamaldehyde sensation in C. elegans."

Ronan, E.A., Guo, Y., Sartbaeva, E., Zhang, X., Liu, J., Xu, X.Z.S., He, F.

[International Worm Meeting, 2019]

Cinnamaldehyde (CA), the essential oil in cinnamon, is shown to have systemic metabolic benefits in both mice and humans, however the underlying molecular mechanisms are not understood. While the only known receptor for CA is transient receptor potential (TRP)A1, the thermogenic response of adipocytes to CA are TRPA1-independent, indicating unknown CA-sensing mechanisms exist. Here we report that CA acts on sensory neurons in C. elegans to evoke complex behavioral responses. In particular, we found that CA can excite the polymodal nociceptive neuron ASH shown by calcium imaging and electrophysiology recordings. We found these effects to be mediated by GPCR signaling, suggesting that the underlying CA receptor is a GPCR. We performed genetic screens to identify the genes mediating CA-avoidance in the ASH neuron. Our results show that C. elegans sense CA through a previously uncharacterized neural and genetic pathway which may be evolutionarily conserved and have physiological significance in higher organisms.

Kouichi Iwasaki et al. (2003) International Worm Meeting "Development of a new assay system to study intracellular calcium dynamics using the C. elegans intestine."

Kouichi Iwasaki, Takayuki Teramoto

[International Worm Meeting, 2003]

The free cytosolic calcium concentration ([Ca]i) regulates various cellular functions and is tightly regulated in the parameters of time, space, and amplitude. When a cell is given a single dose of stimulus within the physiological range, the cell often generates a repetitive increase/fall cycle of intracellular Ca concentration (Ca oscillation). Also, the [Ca]i increase in a single cell travels as a Ca wave through neighboring cells to coordinate their activities. These [Ca]i regulations are ubiquitously observed in various cell types of many organisms. The C. elegans defecation is a periodic behavior and its activity can be used to monitor the [Ca]i regulation in intestine: the worm defecates every 45 seconds and this periodicity is regulated by the activity of the inositol 1,4,5-trisphosphate receptor that is expressed in the intestine (Dal Santo et al. 1999). To visualize cytoplasmic Ca dynamics in the intestine, we expressed the Ca-indicating protein Cameleon. A Ca spike coincides with the first step of the defecation motor program (pBoc), therefore, the [Ca]i oscillates every 45 seconds. The [Ca]i change was between 100 to 600 nM during the pBoc. This [Ca]i oscillation is initiated at the posterior end and is propagated toward the anterior end. Since the C. elegans intestine consists of 20 cells, this system can also be a model to study the intercellular Ca wave propagation. To make this assay system more useful for physiological study, we have developed a new culture system of the C. elegans adult intestine. Christensen et al. (2002) developed C. elegans embryonic cells recently, but our system is distinct and has unique advantages for the study of intestinal physiology. In this system, we can reproduce a variety of phenomena observed in the intact adult intestine, such as execution of pBoc, the intracellular Ca oscillation, and the intercellular Ca wave propagation. The Ca oscillation was also observed using the chemical Ca indicator Fluo-4 and electrophysiological measurements. (The intestinal membrane potential oscillation was first observed by A. Wei.) Therefore, this new system can combine genetic, molecular-biological, and physiological approaches to study a Ca signaling mechanism. Using this assay system, we have investigated function of the Transient Receptor Potential (TRP) Channels that are proposed to be involved in intracellular Ca dynamics, and will discuss their function and the usefulness of this new system for physiological study.

Daisuke Kanematsu et al. (2002) Japanese Worm Meeting "Is IP3 Receptor or Ryanodine Receptor Involved in a Transient Increase of Cytosolic Ca2+ - Concentration during Fertilization of C. elegans Oocyte?"

Yasuji Sakube, Hideyo Kuroda, Hiroaki Kagawa, Ritsu Kuroda, Daisuke Kanematsu

[Japanese Worm Meeting, 2002]

Transient increases of intracellular calcium ion concentration ([Ca 2+ ] i ) have been observed during fertilization of eggs, from invertebrates to mammals. Caenorhabditis elegans seems to be a hopeful model organism to study the signaling pathway from a sperm-egg fusion to a transient increase in [Ca 2+ ] i . Since we have not succeeded to bring about the fertilization in vitro , we measured the [Ca 2+ ] i changes of oocytes during fertilization in vivo . A Ca 2+ indicator, Calcium-Green 1 dextran (10,000 MW) was injected into the distal portion of the ovary of adult worms. Two hrs after injection, the oocytescontaining the indicator lined up at the proximal portion of the ovary. Even in the worm anesthetized with tricaine and tetramisole, the oocytes moved against the proximal end of the ovary and entered into spermatheca in order. We collected the [Ca 2+ ] i images of oocytes in the ovary or spermatheca with a cooled CCD camera. We observed the fluorescent change of hermaphrodite oocytes during self-fertilization. Several seconds after passing through the valve between the ovary and spermatheca, an oocyte showed a transient increase in [Ca 2+ ] i . These results suggest that sperm of C. elegans fertilizes oocytes in the spermatheca and bring about a transient change in [Ca 2+ ] i of oocytes. In mammal eggs, the inositol 1,4,5-trisphosphate receptor/channels (IP 3 R) of endoplasmic reticulum membrane are responsible for these Ca 2+ -changes. The Ca 2+ -transients of sea urchin eggs seems to depend on the synergistical release via both of IP 3 R and ryanodine receptor/channel (RyR). To examine the contribution of IP 3 R or RyR to the Ca 2+ -transient of C. elegans , we examined the changes in [Ca 2+ ] i during self-fertilization of mutant strains of two genes, unc-68 encoding RyR and itr-1 encoding IP 3 R. Both in oocytes of unc-68 and itr-1 , similar changes to the Ca 2+ -transient in N2 oocytes were recorded. These results suggest two possibility, the Ca 2+ -transient during fertilization depend on (1) the synergistical release of Ca 2+ via RyR and IP 3 R from endoplasmic reticulum, or (2) the Ca 2+ -influx through a Ca 2+ channel of the plasmamembrane. To examine these possibilities, we are trying the RNAi of itr-1 and unc-68 .

Laura Bianchi et al. (2004) East Coast Worm Meeting "Calcium permeability of death-inducing DEG/ENaC ion channel MEC-4(d)"

Jian Xue, Maryanne Royal, Laura Bianchi, Beate Gerstbrein, Wei-Hsiang Lee, Monica Driscoll, Gargi Mukherjee, Dewey Royal

[East Coast Worm Meeting, 2004]

The C. elegans ion channel complex formed by the DEG/ENaC MEC-4 and MEC-10 subunits, exclusively assembled in six specialized touch sensing neurons, is thought to constitute the core of a voltage-independent Na + -selective mechanosensory ion channel. In a screen for touch insensitive nematodes, a very interesting mec-4 mutant was isolated. This mec-4(d) mutation specified a large side-chain amino acid substitution near the channel pore and induces degeneration of touch neurons through a mechanism that appears to involve channel hyperactivation . Analysis of mec-4(d)- induced neurodegeneration , however, indicated that large increase of [Ca 2+ ] i is one of the molecular requirements for cell death. While Ca 2+ release from ER-based stores is certainly occurring in mec-4(d) -induced cell-death, the triggering factor remains unknown. We speculated that Ca 2+ could permeate through MEC-4(d) channels to perhaps induce Ca 2+ -induced Ca 2+ release from stores. P> P> We analyzed Ca 2+ permeability of MEC-4(d) channels reconstituted in Xenopus oocytes and found that they are permeable to Ca 2+ ( P Ca /P Na ~ 0.22). Interestingly, Ca 2+ currents through MEC-4(d) channels display the same sensitivity to amiloride that Na + currents do, with Ki for the drug being reduced by co-expression with MEC-10(d). We also found that Ca 2+ permeability is independent of co-expression with MEC-10(d), MEC-2 or MEC-6, and appears to be determined by MEC-4(d) residues. We are currently searching for residues involved in Ca 2+ permeability, by introducing second site mutations in and near the pore, identified in our screen for suppressors of mec-4(d) -induced cell death. In addition we are evaluating the intracellular pathway activated by entry of Ca 2+ through MEC-4(d) that leads to massive release of Ca 2+ from the stores that ultimately results in cell death.

Laura Bianchi et al. (2004) West Coast Worm Meeting "Calcium permeability of death-inducing DEG/ENaC ion channel MEC-4(d)"

Dewey Royal, Monica Driscoll, Beate Gerstbrein, Gargi Mukherjee, Jian Xue, Laura Bianchi

[West Coast Worm Meeting, 2004]

The C. elegans ion channel complex formed by the DEG/ENaC MEC-4 and MEC-10 subunits, exclusively assembled in six specialized touch sensing neurons, is thought to constitute the core of a voltage-independent Na + -selective mechanosensory ion channel. In a screen for touch insensitive nematodes, a very interesting mec-4 mutant was isolated. This mec-4(d) mutation specified a large side-chain amino acid substitution near the channel pore and induces degeneration of touch neurons through a mechanism that appears to involve channel hyperactivation . Analysis of mec-4(d)- induced neurodegeneration , however, indicated that large increase of [Ca 2+ ] i is one of the molecular requirements for cell death. While Ca 2+ release from ER-based stores is certainly occurring in mec-4(d) -induced cell-death, the triggering factor remains unknown. We speculated that Ca 2+ could permeate through MEC-4(d) channels to perhaps induce Ca 2+ -induced Ca 2+ release from stores. P> P> We analyzed Ca 2+ permeability of MEC-4(d) channels reconstituted in Xenopus oocytes and found that they are permeable to Ca 2+ ( P Ca /P Na ~ 0.22). Interestingly, Ca 2+ currents through MEC-4(d) channels display the same sensitivity to amiloride that Na + currents do, with Ki for the drug being reduced by co-expression with MEC-10(d). We also found that Ca 2+ permeability is independent of co-expression with MEC-10(d), MEC-2 or MEC-6, and appears to be determined by MEC-4(d) residues. We are currently searching for residues involved in Ca 2+ permeability, by introducing second site mutations in and near the pore, identified in our screen for suppressors of mec-4(d) -induced cell death. In addition we are evaluating the intracellular pathway activated by entry of Ca 2+ through MEC-4(d) that leads to massive release of Ca 2+ from the stores that ultimately results in cell death.

V Centonze et al. (1999) International C. elegans Meeting "Excitation-contraction coupling in C. elegans muscle cells as monitored by multiphoton excitiation FRET imaging of calcium indicator proteins."

B Saari, V Centonze, P Anderson, D Wokosin, E Maryon

[International C. elegans Meeting, 1999]

Muscle contraction is initiated by membrane depolarization followed by a rapid elevation of cytoplasmic Ca 2+ . This process, called excitation-contraction coupling, is mediated by voltage gated Ca 2+ channels in the plasma membrane and intracellular Ca 2+ channels in the sarcoplasmic reticulum. Using a system similar to that described by Kerr, et al. (WCWM '98) we are characterizing Ca 2+ transients during pharyngeal pumping in wild-type animals, which we will compare to transients in animals having mutations in genes such as Ca 2+ channels. Multiphoton excitation is being used to elicit Ca 2+ -dependent fluorescence resonance energy transfer (FRET) emissions from a cameleon (ycam2) protein (provided by R. Tsien). ycam2 consists of two mutant GFP moieties having differing excitation and emission spectra, connected by a Ca 2+ -binding linker peptide. When ycam2 binds Ca 2+ it undergoes a conformational change that allows FRET between the 480nm-emitting moiety (cyan fluorescent protein-CFP) and the 535nm-emitting moiety (yellow fluorescent protein-YFP). Ca 2+ increases are thus detectable as an increase in the YFP/CFP emission ratio. We have expressed ycam2 in pharyngeal muscle cells using the myo-2 promoter. Imaging is performed on a prototype multidimensional multiphoton microscope. The instrument allows wide field direct detection of 3 channels: 1) bright field image; 2) YFP emission (535-550nm); 3) CFP emission (380-485nm). ycam2 fluorescence is imaged with a Ti-sapphire laser tuned to 800nm to primarily excite the CFP moiety. Time series are collected and data is subsequently analyzed using NIH-image. We have observed Ca 2+ transients during pharyngeal pumping by dissecting pharynxes in serotonin containing saline and imaging the terminal bulb. We observe 10% changes in YFP/CFP emission ratio from "baseline" to "peak" which correlate with opening of the terminal bulb lumen. Although our system does not yet allow "real time" resolution of Ca 2+ transients, we can correlate pharyngeal muscle contraction with calcium elevation in wild-type and mutant animals. This work was supported by RR00570.

Zahratka, Jeffrey et al. (2013) International Worm Meeting "Multiple independent calcium pools in a nociceptive neuron in C. elegans."

Bamber, Bruce, Komuniecki, Richard, Zahratka, Jeffrey, Williams, Paul

[International Worm Meeting, 2013]

To navigate constantly changing environments, animals integrate sensory inputs and internal state information to formulate proper locomotory commands. Monoamines and neuropeptides encode internal state information, modulating sensorimotor circuits at several levels, including the sensory neurons themselves. We study aversive behaviors in C. elegans to better understand how neuromodulators affect circuit function, in terms of the physiological states of single identifiable neurons. C. elegans reacts to the noxious odorant 1-octanol by reversing locomotory direction, to move away from the odor source. Worms off food respond to diluted 1-octanol in 10s, but in the presence of food, response time shortens to 5s. Endogenous 5-HT and neuropeptides are key signaling intermediates in this food-dependent modulation, relying in part on a 5-HT receptor (SER-5) in the ASHs (the pair of nociceptive neurons that sense 1-octanol). Using the GCaMP3 Ca++ indicator, we show that a saturating concentration of 1-octanol dissolved in buffer (~2 mM) elicits robust excitation of ASHs when applied to the nose. At least 3 distinct Ca++ pools exist in ASHs: distal dendrite, proximal dendrite/soma, and axon. Proximal dendrite/soma Ca++ responses are sensitive to Nemadipine-A, a blocker of L-type Ca++ channels. Distal dendrite responses, and more importantly, axonal responses are resistant to Nemadipine-A. This finding suggests that somal Ca++ influx is unnecessary for signal propagation from one end of the cell to the other, consistent with isopotentiality within C. elegans sensory neurons. What then is the role of somal Ca++ influx? Rather than amplifying or relaying chemosensory signals to axons, we hypothesize it is intermediate in sensory modulation, and in support, monoamine signals regulating aversive behavior also regulate ASH somal Ca++ signals. We are currently testing whether the different Ca++ pools within ASHs are the result of regionalized expression of Ca++ channels, characterizing the monoamine and neuropeptide signaling cascades affecting ASH somal Ca++, and elucidating the roles for ASH somal Ca++ by direct measurement of ASH synaptic release onto postsynaptic targets and behavioral assays.

Takayuki Teramoto et al. (2004) West Coast Worm Meeting "Molecular cloning and physiological characterization of the defecation rhythm controlling gene dec-1"

Kouichi Iwasaki, Takayuki Teramoto

[West Coast Worm Meeting, 2004]

The C. elegans defecation is a rhythmic behavior: with abundant food, a defecation motor program (DMP) is activated every 45 seconds. It was reported that this defecation rhythm is regulated by the inositol 1,4,5-trisphosphate (IP3) receptor in the intestine (Dal Santo et al., 1999). Therefore, the defecation phenotype can be used to monitor activities of an IP3-dependent Ca ++ -signaling pathway. To visualize cytoplasmic Ca ++ dynamics in the C. elegans intestine, we expressed the Ca ++ -indicating protein, cameleon. The first step of the defecation motor program (the posterior body wall muscle contraction, pBoc) coincided with a Ca ++ spike. This Ca ++ spike was initiated at the posterior end of the intestine and propagated through the entire intestine toward the anterior end. To make this imaging system more useful for physiological study, we have developed a culture system of the C. elegans adult intestine. In this system, we can reproduce a variety of phenomena observed in the intact adult intestine, such as execution of a defection motor program step, Ca ++ oscillation, and Ca ++ -wave propagation. We have also adapted Ca ++ imaging using the chemical indicators Fluo-4 and Fura Red for the dissected intestinal preparations. Fluo-4 increases its fluorescence intensity while Fura Red decreases when [Ca ++ ] increases, therefore, the ratiometric measurement using these indicators maximizes the detection sensitivity and is less likely to pick up false signals. To elucidate the regulatory mechanism of this rhythm generation, we are molecularly cloning dec-1 . The dec-1 mutant was originally isolated by J.H. Thomas based on a constipated phenotype and shows a prolonged DMP rhythm. Along with molecular cloning, we are currently investigating if the intestinal Ca ++ dynamics is altered in the dec-1 mutant. Dal Santo et al . (1999) Cell 98: 757-767 Thomas. (1990) Genetics 124: 855-872

Edgley ML et al. (1997) International C. elegans Meeting "Genetic Toolkit: Progress on Chromosome II"

Edgley ML, Riddle DL, Turner CA

[International C. elegans Meeting, 1997]

We are building a genetic toolkit for the C. elegans genome, consisting of regional chromosomal balancers and sets of overlapping balanced deficiencies whose endpoints will be tied to the physical map. In recent work we have concentrated on making new balancers for the left arm of LG II, in the interval to the left of dpy-10. New balancers including mC4, mC5, mC6, and mT1 (previously called mC7) were isolated and partially characterized. All suppress recombination in the interval between rol-1 and unc-85, and all are marked with dpy-10(e128). mT1 has been shown to be a II;III translocation, causing pseudolinkage of markers from the right arms of both chromosomes (rol-1 II and unc-71 III). Its left breakpoint on LG II apparently lies very near dpy-10, and it thus does not balance well in our region of interest. However, it is a potential alternative to the balancers in common use, mnC1(II) or eT1(III;V). mC5 and mC6 both balance well, reducing recombination between rol-1 and unc-85 from about 8.5 map units to about 0.2 map units. Of the two, mC6 is much more stable. It balances extremely well from rol-1 (the rightmost marker tested so far) left to let-552, although suppression of recombination falls off sharply between let-552 and sqt-2. We are now using mC6 to isolate and map deficiencies. Approximately 3000 chromosomes mutagenized with 0.07% formaldehyde were screened, resulting in three balanced lethals. We recently switched to UV mutagenesis. Once we have a number of balanced deficiencies in hand, we plan to locate their endpoints, designing PCR primers from DNA sequence generated by the C. elegans genome project. This work is funded by the NIH National Center for Research Resources (NCRR).

Ed Maryon et al. (2001) International C. elegans Meeting "Calcium Regulatory Sites in UNC-68 Ryanodine Receptor Channels"

Ed Maryon, Phil Anderson, Bonnie Saari, Amy Riek, LuAnne Sowinski

[International C. elegans Meeting, 2001]

Ryanodine receptors (RyRs) are intracellular, homotetrameric ion channels in muscle cells that gate the release of calcium (Ca 2+ ) from the sarcoplasmic reticulum (SR). RyR channels flood the myoplasm with Ca 2+ following depolarization of the muscle surface membrane, causing contraction. RyRs are regulated by myoplasmic Ca 2+ . Depolarization is accompanied by a modest influx of Ca 2+ through voltage-gated Ca 2+ channels, which activates RyR channels. Subsequently, RyRs are inactivated by the higher Ca 2+ levels present after SR Ca 2+ -release, thus creating a self-terminating burst of Ca 2+ during muscle contraction. C. elegans has a single RyR gene ( unc-68) that encodes a 5074 amino acid RyR protein. In body-wall muscle cells, UNC--68 channels are localized in SR vesicles between the surface membrane and the myofilament lattice. unc--68 null mutant animals are viable, but have defective locomotion. We are examining the biological role of Ca 2+ binding sites in UNC-68 RyRs that may be involved in activation or termination of Ca 2+ release in body-wall muscle cells. The predicted UNC-68 protein has two EF hand-type Ca 2+ -binding motifs at amino acids 4198-4246 (Sakube, et. al. 1997 JMB 267 :849). We have eliminated one or both EF hands using alanine substitution, and analyzed 45 Ca 2+ -binding to wild type and mutant fusion proteins containing the EF-hand region. Both the EF hand motifs bind 45 Ca 2+ , as shown by 45 Ca 2+ -overlays and equilibrium binding. We have expressed GFP-tagged unc-68 genes containing wild-type EF-Hands or the EF-Hand mutations in unc--68 null mutant animals. Proper expression and localization of the GFP-tagged RyRs have been confirmed using in vivo localization of the GFP tag, and western blot analysis of purified, detergent-solubilized RyR proteins. Both intact EF hands are required for rescue of motility (i.e. only the WT gene rescues normal locomotion). The two genes with a single EF hand eliminated (EF1 + EF2 - or EF1 - EF2 + ), give partial rescue of motility, and restore sensitivity to paralysis by ryanodine. The gene lacking both EF-Hand calcium-binding sites does not rescue motility or restore sensitivity to ryanodine. Interestingly, when expressed in unc-68(0)/+ or wild-type animals, arrays containing all three EF-Hand mutant RyR genes exert semi-dominant effects on motility. One interpretation of this semi-dominance is that the mutant RyR subunits form heterotetramers with Wild Type subunits. Further characterization of the purified mutant RyR channels should reveal whether the EF-Hand calcium-binding sites are involved in activation or inactivation of calcium release during body-wall muscle contraction.