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[
Curr Biol,
2002]
The phasmids are bilateral sensory organs located in the tail of Caenorhabditis elegans and other nematodes. The similar structures of the phasmids and the amphid chemosensory organs in the head have long suggested a chemosensory function for the phasmids [1]. However, the PHA and PHB phasmid neurons are not required for chemotaxis [2, 3] or for dauer formation [4], and no direct proof of a chemosensory function of the phasmids has been obtained. C. elegans avoids toxic chemicals by reversing its movement, and this behavior is mediated by sensory neurons of the amphid, particularly, the ASH neurons [5, 6]. Here we show that the PHA and PHB phasmid neurons function as chemosensory cells that negatively modulate reversals to repellents. The antagonistic activity of head and tail sensory neurons is integrated to generate appropriate escape behaviors: detection of a repellent by head neurons mediates reversals, which are suppressed by antagonistic inputs from tail neurons. Our results suggest that C. elegans senses repellents by defining a head-to-tail spatial map of the chemical environment.
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[
European Worm Meeting,
2006]
Chemical sensitivity allows animals to identify and respond appropriately to the chemical composition of the environment. Noxious water-soluble compounds that are avoided by C. elegans are generally sensed as bitter by humans and are discarded in double choice test by mice. We have used C. elegans to focus on the molecular mechanisms involved in primary sensing of quinine, a molecule detected as bitter by humans. ASH is the main sensory neuron involved in sensing quinine. Two G? subunits, GPA-3 and ODR-3 are necessary for the response of ASH to repellent stimuli (Hilliard et al., 2004 and 2005). In addition the TRPV channel proteins, OSM-9 and OCR-2, are also necessary for the ASH avoidance responses (Colbert et al 1997, Tobin et al 2002). Finally we identified a novel protein, QUI-1, as an essential components of the response to quinine (Hilliard et al., 2004). With regard to the molecular function of QUI-1, we demonstrate that QUI-1 function is required in ASH for the response to quinine and, using specific antibodies, that the protein is localized to the sensory cilia. These results, together with the discovery that QUI-1 contains an RGS (Regulator of G protein Signaling) domain, strongly suggest that this novel protein might be involved in quinine signaling.. Are there other components of the quinine signal transduction pathway?. We are using a best candidate approach and a variety of behavioral assays to identify new molecules involved in sensing repellent chemicals and in particular quinine. We analyzed behaviorally loss of function and overexpression mutants in several molecules known to act in the G protein signaling pathways (G? subunits, G? subunits, RGS proteins, etc.). The results obtained will be discussed.. Colbert, H. A., Smith, T. L. and Bargmann, C. I. (1997). OSM-9, a novel protein with structural similarity to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis elegans. J Neurosci 17, 8259-69.. Hilliard, M. A., Apicella, A. J., Kerr, R., Suzuki, H., Bazzicalupo, P. and Schafer, W. R. (2005). In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents. Embo J 24, 63-72.. Hilliard, M. A., Bergamasco, C., Arbucci, S., Plasterk, R. H. and Bazzicalupo, P. (2004). Worms taste bitter: ASH neurons, QUI-1, GPA-3 and ODR-3 mediate quinine avoidance in Caenorhabditis elegans. Embo J 23, 1101-11.. Tobin, D., Madsen, D., Kahn-Kirby, A., Peckol, E., Moulder, G., Barstead, R., Maricq, A. and Bargmann, C. (2002). Combinatorial expression of TRPV channel proteins defines their sensory functions and subcellular localization in C. elegans neurons. Neuron 35, 307-18.
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[
International Worm Meeting,
2013]
EFF-1 and AFF-1 are highly efficient fusogens known to mediate cell-cell fusion events during the development of different C. elegans tissues. Both fusogens are also expressed in neurons, and EFF-1 has recently been shown to play a role in the development and remodelling of neurites1,2, indicating that cell membrane fusion is an important neuronal event in C. elegans. However, very little is known about the molecular mechanisms underpinning membrane fusion within these cells; importantly, it is also unclear how this process is highly restricted to the individual cell, with fusion between neurites of adjacent neurons almost never observed. In non-neuronal tissues where cell-cell fusion occurs, numerous transcription factors and regulators have been identified as being critical for proper cell-cell fusion3. Our hypothesis is that similar mechanisms are in place in neurons, controlling fusion of neurites within individual cells and preventing neurites of adjacent neurons from fusing. We used different pairs of tightly associated neurons, AWCR/AWCL (head) as well as PLM/PLN (tail), to test this hypothesis and to study EFF-1 and AFF-1-mediated fusion in neurons. Using transgenic strains and microscopy techniques, we have found that overexpression of EFF-1 or AFF-1 under neuron-specific promoters leads to mixing of cytoplasms between individual neurons. We confirmed that the cytoplasms are indeed connected by using the photoconvertible protein Kaede. We also determined the temporal requirement of the fusogen by using a construct where EFF-1 is under a heat-shock promoter. In addition, using a fluorescent-tagged version of EFF-1, we are characterising the site of fusion between adjacent neurons when the fusogen is overexpressed. Finally, we have started a genetic screen using neuronal-specific RNAi, where genes known as cell-cell fusion regulators are investigated for their capacity to induce mixing of cytoplasms between associated neurons. The results presented here will give us new insights into the molecular mechanisms that control membrane fusion during development and remodelling of C. elegans neurons. 1Oren-Suissa et al., Science, 2010, 2Gosh-Roy et al., J. Neurosci. 2010, 3Podbilewicz, Wormbook, 2006.
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[
Neuronal Development, Synaptic Function and Behavior, Madison, WI,
2010]
We fabricated and tested a high-throughput microfluidic platform to study nerve regeneration in C. elegans. The device consists of an array of small chambers in a parallel fluidic circuit allowing for simultaneous trapping of dozens of C. elegans worms in individual visualization chambers for in-vivo imaging and laser ablation of fluorescently labeled axons. With proper liquid nutrients, the animals can easily survive in the microfluidic chambers for three days or more for monitoring nerve regeneration. This device could serve as the optical and fluidic interface for automated genome-wide nerve regeneration studies using femtosecond laser nano-axotomy and fluorescence microscopy. Using our device and conventional methods, we investigated the regenerative capacity of the oxygen sensory neuron, PQR. This neuron is located in the left lumbar ganglion on the posterior-lateral side of the worm's body, and has only two processes emerging from the cell body – a dendrite extending posterior toward the tip of the tail and an axon extending anterior joining the ventral nerve cord. We looked at regeneration rates in animals in which either only one or both neurites were severed. We observed that the dendrite process regenerated with a higher frequency when the axon was simultaneously severed. This result suggests that the molecular machinery responsible for regeneration is more efficiently recruited in a given process when there is additional damage to other parts of the neuron.
-
[
Neuronal Development, Synaptic Function and Behavior, Madison, WI,
2010]
Neurons exhibit distinct morphological domains, axons and dendrites, which are essential for functional wiring of the nervous system. While many molecules involved in axon development have been discovered, there is little known about the ligands and receptors that regulate dendrite development. To understand how dendrites develop in C. elegans we focused on the PQR oxygen sensory neuron. PQR has its cell body positioned in the left lumbar ganglion on the posterior-lateral side of the body. A single dendrite extends posterior with sensory cilia at its tip, while the axon extends anterior along the ventral nerve cord. PQR is born post-embryonically allowing easy visualization of dendrite development using the
gcy-36::GFP transgene. In a genetic screen for dendrite defective mutants we isolated a previously uncharacterized mutation in
lin-17, a C. elegans Frizzled receptor gene. We found that in
lin-17(
vd002), the PQR dendrite was absent, shortened or misrouted anterior. Similar dendrite defects were also observed in other known alleles of
lin-17. Cell-specific expression of wild-type LIN-17 in PQR indicated a non-cell-autonomous role of this molecule in regulating dendrite development. LIN-44 is a Wnt ligand known to bind the Frizzled receptor LIN-17 and is expressed by four hypodermal cells in the tip of the tail. We found that
lin-44 mutants presented PQR dendrite defects similar to those observed in
lin-17 mutants. We expressed LIN-44 ectopically from more anterior regions of the body and found that it rescued the PQR dendrite defects of
lin-44 mutants, indicating LIN-44 functions as a permissive cue. Using a heat-shock promoter to drive LIN-44 we determined that the presence of this ligand at the time of PQR dendrite formation was sufficient to rescue the dendrite defects of the
lin-44 mutant. Analysis of the
lin-17 lin-44 double mutant indicated a genetic interaction between these molecules. Our studies provide the first direct evidence that specific Wnt signals and Frizzled receptors regulate dendrite formation in vivo. We propose a model in which PQR dendrite formation is achieved by the interaction of LIN-44 and LIN-17 acting on PQR through its neighbouring cells.
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[
Biochemistry,
2012]
Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.
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[
International Worm Meeting,
2019]
Distinct cell types adhere to each other to form functional systems in an organism. Specifically, in vertebrates, ensheathing glia attach to axons forming a functional unit that responds to injury. In C. elegans, the posterior lateral mechanosensory (PLM) neurons have an intimate relationship with their surrounding epidermis. During development, the PLM axon becomes ensheathed within the overlying epidermis and is mechanically coupled to this tissue via specialized trans-epidermal attachment structures. The transmembrane protein LET-805/Myotactin is a component of these attachment structures, and is proposed to be required for correct attachment of the mechanosensory neuron axon to the epidermis as well as for the attachment of the epidermis to the body wall muscles. We visualized the localisation of LET-805, and attachment sites, using a CRISPR/Cas9 engineered C-terminal wrmScarlet tag in a wild-type background, together with a PLM neuron-specific cytosolic GFP marker. To characterize the role of neuronal attachment in axonal maintenance and repair after axonal injury, we axotomized the PLM neuron using UV-laser and visualized LET-805::wrmScarlet before, during, and after injury. In uninjured animals at the L4 stage, LET-805::wrmScarlet localized to periodic puncta over the PLM axon. After injury, we observed that LET-805::wrmScarlet was slowly lost in regions corresponding to the disconnected distal axon fragment of PLM, often occurring after the loss of any visible cytosolic neuronal marker. On the regrowing proximal axon, we observed that LET-805::wrmScarlet did not localize to the newly regrowing axons for at least 48 hours, after which it reassembled into puncta following the path traced by the regrowing axon. Taken together, our data suggests that following injury the attachment of the PLM neuron to its surrounding tissue is maintained and is not highly dynamic. We propose that the regrowing axonal fragment induces re-attachment to the epidermis. We are currently testing whether modulation of axonal attachment impacts axonal degeneration or regeneration in this system, and how injuries on PLM axon are detected by the surrounding epidermis.
-
[
Worm Breeder's Gazette,
1994]
cej-1 Encodes a Novel Protein with Poly-Threonine Motif M. L. A. Khanl, M. Tabish, T. Fukushigel1 S. Tsukita2, M. Itoh , Sh. Tsukita , and S. S. Siddiqui. (1): Lab. of Molecular Biology, Dept of Ecological Engg. Toyohashi Univ. Technology, Toyohashi 441, and (2). National Institute for Physiological Sciences, Okazaki 444, Japan.
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[
Worm Breeder's Gazette,
1976]
We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.
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[
Mech Ageing Dev,
2009]
Energy production via oxidative phosphorylation generates a mitochondrial membrane potential (DeltaPsi(m)) across the inner membrane. In this work, we show that a lower DeltaPsi(m) is associated with increased lifespan in Caenorhabditis elegans. The long-lived mutants
daf-2(
e1370),
age-1(
hx546),
clk-1(
qm30),
isp-1(
qm150) and
eat-2(
ad465) all have a lower DeltaPsi(m) than wild type animals. The lower DeltaPsi(m) of
daf-2(
e1370) is
daf-16 dependent, indicating that the insulin-like signaling pathway not only regulates lifespan but also mitochondrial energetics. RNA interference (RNAi) against 17 genes shown to extend lifespan also decrease DeltaPsi(m). Furthermore, lifespan can be significantly extended with the uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP), which dissipates DeltaPsi(m). We conclude that longevity pathways converge on the mitochondria and lead to a decreased DeltaPsi(m). Our results are consistent with the 'uncoupling to survive' hypothesis, which states that dissipation of the DeltaPsi(m) will extend lifespan.