Koichi Hasegawa et al. (2004) East Asia Worm Meeting "Acrylamide effects on the nematode Caenorhabditis elegenans"

Satsuki Miwa, Johji Miwa, Hajime Taniguchi, Kaname Tsutsumiuchi, Koichi Hasegawa

[East Asia Worm Meeting, 2004]

The neurotoxic, genotoxic and carcinogenic effects of industrial exposure to acrylamide (AA) have been studied in animals and humans for more than 30 years. A recent search for the cause of high background levels of AA in industrially unexposed people revealed that it is formed during the frying or baking of foods by means of the Maillard reaction. To evaluate the biological consequences of continuous exposure to AA at levels found in common foodstuffs, we studied the effects of AA on growth, fecundity and lifespan in the nematode Caenorhabditis elegans . No deleterious consequences are observed for growth or fecundity when the animals are raised in the AA concentrations of 0.5ug/L to 5mg/L, which are commonly found in daily consumed foodstuffs. Conversely, in 500mg/L of AA, they show retarded growth with reduced body and brood sizes. Unlike the first two parameters, however, lifespan is decreased significantly even in 0.5ug/L of AA. Furthermore, the AA effect on lifespan is biphasic as lifespan is increased to near normalcy in 5mg/L and decreased again in 500mg/L.Next we examined the gene expression profiles of C. elegans grown on two AA concentrations, extremely low concentration (0.5ug/L) and high concentration (500mg/L), by using the Stanford Microarray Technology. Of the 21,120 genes profiled, 14 genes are up and 56 genes are down regulated in 0.5ug/L AA. In 500mg/L AA, 438 genes are up and 344 genes are down regulated. Of the 438 up-regulated genes, 32 genes code for members of the glutathione S-transferases, supposed to be the main detoxicants of AA, and 34 for major sperm proteins. C. elegans provides an excellent model system for examining and evaluating AA toxicity on the whole animal level; and the biphasic nature of the dose response exhibited by AA in the lifespan warns us to re-evaluate the present risk assessment of environmental pollutants and harmful substances in foods.

BT Tsung et al. (1999) International C. elegans Meeting "Adapting a manual clonal screen to semi-automation"

PB Krauledat, WP Hansen, AA Ferrante, C Johnson, BT Tsung, CP Hunter

[International C. elegans Meeting, 1999]

Clonal screens are an effective, but labor intensive, means to identify mutants with specific phenotypes. We are exploring the capabilities of a nematode flow-sorting system to assist with our continuing clonal screen for conditional lethal mutations (see abstract by Tsung et al., this meeting). The system (developed by Union Biometrica, Somerville MA. in collaboration with Axys, Nemapharm group) automatically analyzes and sorts individual nematodes by length (indicative of development stage or mutant type) from mixed populations and distributes them, along with a minimal volume, unharmed into microplates. Individual F2 L4 worms from a synchronized population were deposited into each well of a 96-well micro-titer culture plate seeded with 50 microl of diluted HB101. The current system accurately delivered a single nematode of the selected size (in our case 15% of the heavily mutagenized population were of the selected size) into 95/96 wells on 82 plates at about 3 minutes per plate. All worms were viable in test runs using non-mutagenized worms. Comparisons to our manual screen will be presented.

M Pilon et al. (1999) International C. elegans Meeting "C32E8.7, a C.elegans homolog of the diabetes autoantigen ICA69"

M Pilon, HM Dosch, AM Spence

[International C. elegans Meeting, 1999]

Type I diabetes is an autoimmune disease in which the immune system targets antigens in the insulin-producing pancreatic beta-cells. The Islet Cell Antigen of 69 Kd (ICA69) was discovered by screening islet cell expression libraries with serum from diabetic rats or human patients. The protein exhibits no significant homology with proteins of known function, except for the presence of a putative coiled coil motif possibly implicated in protein-protein interactions. Based on its amino acid sequence, ICA69 is predicted to be a cytosolic soluble protein. Only one ICA69 gene seems to exist in mouse (478 aa), rat (480 aa) and human (483 aa); these exhibit 87% overall identity. In mouse, the ICA69 protein is expressed in most organs, but is most abundant in tissues rich in neuroendocrine or secretory cells such as brain, pancreas and stomach mucosa. The C32E8.7 (418 aa) predicted gene encodes a C.elegans homolog of ICA69 (33% overall identity with human ICA69, including a stretch of 280 aa with 58% conservation). Using a GFP reporter in transgenic C.elegans , we found that the C32E8.7 promoter is specifically expressed in all C.elegans neurons; the timing of promoter activity coincides with the appearance of differentiated neurons during embryogenesis (~400 minutes). Because: 1) most diabetes autoantigens are components of the insulin secretory granules; 2) The high levels of ICA69 expression in neuroendocrine/secretory tissues; and 3) the C.elegans homolog of ICA69 is expressed in differentiated neurons, we hypothesized that ICA69 is involved in regulated exocytosis which mediates, for example, insulin secretion by pancreatic beta-cells and neurotransmitter release by neurons. In collaboration with Prof. Plasterk (Amsterdam, NL), we have isolated a C32E8.7 mutant (NL2003) in which nucleotides -395 to +2276 (262 codons) have been deleted. The NL2003 mutant exhibits no overt phenotype. However, we have observed a slight shortening of life span, and preliminary experiments suggest that the mutant is slightly resistant to aldicarb, an inhibitor of acetylcholinesterase. This later observation would support our hypothesis that ICA69, and its C.elegans homolog, is involved in exocytosis.

Lee, Hyeon-Cheol et al. (2009) International Worm Meeting "mboa-7 is required for selective incorporation of polyunsaturated fatty acids into phosphatidylinositol in C. elegans."

Lee, Hyeon-Cheol, Gengyo-Ando, Keiko, Inoue, Takao, Mitani, Shohei, Arai, Hiroyuki

[International Worm Meeting, 2009]

Phosphatidylinositol (PI) is a component of membrane phospholipids functioning both as a signaling molecule and as a compartment-specific localization signal in form of polyphosphoinositides. Arachidonic acid (AA) is the predominant fatty acid in the sn-2 position of PI in mammals. LysoPI acyltransferase (LPIAT) is thought to catalyze formation of AA-containing PI; however, the gene that encodes this enzyme has not yet been identified. In this study, we established a screening system to identify genes required for utilization of exogenous polyunsaturated fatty acids in C. elegans, in which eicosapentaenoic acid (EPA) instead of AA is dominant in PI. We found that a previously uncharacterized gene, which we named mboa-7, is required for incorporation of PUFAs into PI. Incorporation of exogenous EPA into PI of the living worms and the in vitro incorporation of EPA into lysoPI by the microsomal fraction were greatly reduced in mboa-7 mutants while the incorporation of EPA into other phospholipids was unaffected in both systems. siRNA of human mboa-7 homologue (MBOAT7) in HeLa cells also reduced AA incorporation into PI at cellular level and in vitro. Furthermore, the membrane fractions of transgenic worms expressing recombinant MBOA-7 and MBOAT7 exhibited remarkably increased LPIAT activity but not other lysophospholipid acyltransferase activity. Finally, mboa-7 mutants had greatly reduced EPA content in PI but not in other phospholipids. These data demonstrated for the first time that C. elegans mboa-7 and its mammalian homologue MBOAT7 are LPIAT functioning as a determinant of PI molecular species.

Vasquez, V. et al. (2013) International Worm Meeting "Membrane phospholipids that contain arachidonic acid regulate touch receptor neuron mechanics and touch sensation."

Krieg, M., Vasquez, V., Goodman, M.B., Lockhead, D.

[International Worm Meeting, 2013]

Touch, proprioception, and blood pressure regulation rely on the ability of mechano-electrical transduction (MeT) channels to convert mechanical stimuli into electrochemical signals. The protein subunits that form MeT channel complexes are known only in a small set of mechanosensory neurons, including the touch receptor neurons (TRNs) responsible for gentle body touch in C. elegans. Little is known about the interplay between these channel complexes and the surrounding membrane environment. However, three proteins in the complex are believed to bind lipids: MEC-2, UNC-24, and MEC-6. The TRNs are ideal to test the effect of lipids in MeT because electrical responses to touch depend only on one kind of MeT channel (the ASH nociceptors have two MeT channels) and because connections to downstream neurons are well-characterized. We tested a model in which lipids regulate the mechanical response of TRNs by defining the membrane environment around the MeT channel complex. To this end, we combined genetic dissection (fat and mboa mutants) with behavioral studies, chemical complementation, optogenetic, and biophysical approaches to determine the role of membrane lipids in the response to gentle touch. We demonstrate that disrupting arachidonic acid (AA) or its incorporation into phospholipids impairs TRN-dependent behavioral responses. Thus, membrane phospholipids containing AA are critical for touch sensitivity. AA is likely synthesized within TRNs in vivo, since we show that fat-4, the enzyme needed for its synthesis, is expressed in TRNs. We used optogenetics to show that the defect in touch sensation likely reflects a loss of mechanotransduction rather than downstream signaling. Finally, we found that loss of long polyunsaturated fatty acids, including AA, increases bending stiffness of TRN membranes, as determined by atomic force microscopy. Our findings demonstrate that when AA is part of membrane phospholipids it helps to fine-tune the mechanical properties of TRN membranes and is crucial for MeT. These findings provide a framework for understanding eukaryotic mechanotransduction as a process that relies on a cellular machine composed of both proteins and membrane lipids.

CD Johnson et al. (1999) International C. elegans Meeting "Automated sorting and dispensing of C. elegans to wells of microtiter plates"

B Reardon, CD Johnson, PB Krauledat, WP Hansen, AA Ferrante, R Clover

[International C. elegans Meeting, 1999]

For generating a large number of separate populations of worms, liquid cultures in the wells of microtiter plates use space more efficiently than do standard cultures on agar plates. At NemaPharm, we routinely grow worms in microtiter plates for a wide variety of procedures including both forward and reverse genetics experiments as well as for high-throughput chemical screening. To automate distribution of worms into microtiter plate wells, we have constructed a machine that efficiently sorts and dispenses live nematodes. The mechanism used for measuring nematodes is based, in principle, on the nematode counting machine built by Lou Byerly, Randy Cassada and Dick Russell in the early 1970's (1). Nematodes are suspended in liquid and passed though a narrow nozzle into the center of a rapidly flowing sheath current. The resultant shear forces straighten the animals and orient them parallel to the direction of flow. The straightened animals are then passed at high speed (~1 m/sec) through an orthogonal sheet of laser light where, in the current model, an in-line detector measures the attenuation of the laser light and an attached computer calculates the length of the nematode based upon time-of-flight through the beam. Below the detector, the worm-containing stream is deflected to waste by a air jet controlled by the computer that shuts off momentarily to deliver a selected animal to a well (distribution volume is 0.25 - 1 microl per worm). The current sorter/dispenser is capable of distributing 10 worms to the wells of 96-well plates in 2 minutes with a precision of +/-0.7 worms. Distribution to 384 and 1536 well plates is also feasible with the current model. Machines with more sophisticated detecting and measuring capabilities are planned for the future. (1) Byerly, et. al. (1975) Rev Sci. Instrum., 46 :517-522

Hyeon-Cheol Lee et al. (2010) East Asia Worm Meeting "C. elegans mboa-7, a Member of the MBOAT Family, is Required for Selective Incorporation of Polyunsaturated Fatty Acids into Phosphatidylinositol"

Hyeon-Cheol Lee, Hiroyuki Arai, Shohei MItani, Takao Inoue, Keiko Gengyo-Ando

[East Asia Worm Meeting, 2010]

Phosphatidylinositol (PI) is a component of membrane phospholipids functioning both as a signaling molecule and as a compartment-specific localization signal in form of polyphosphoinositides. Arachidonic acid (AA) is the predominant fatty acid in the sn-2 position of PI in mammals. LysoPI acyltransferase (LPIAT) is thought to catalyze formation of AA-containing PI; however, the gene that encodes this enzyme has not yet been identified. In this study, we established a screening system to identify genes required for utilization of exogenous polyunsaturated fatty acids in C. elegans, in which eicosapentaenoic acid (EPA) instead of AA is dominant in PI. We found that a previously uncharacterized gene, which we named mboa-7, is required for incorporation of PUFAs into PI. Incorporation of exogenous EPA into PI of the living worms and the in vitro incorporation of EPA into lysoPI by the microsomal fraction were greatly reduced in mboa-7 mutants while the incorporation of EPA into other phospholipids was unaffected in both systems. siRNA of human mboa-7 homologue (MBOAT7) in HeLa cells also reduced AA incorporation into PI at cellular level and in vitro. Furthermore, the membrane fractions of transgenic worms expressing recombinant MBOA-7 and MBOAT7 exhibited remarkably increased LPIAT activity but not other lysophospholipid acyltransferase activity. Finally, mboa-7 mutants had greatly reduced EPA content in PI but not in other phospholipids. These data demonstrated for the first time that C. elegans mboa-7 and its mammalian homologue MBOAT7 are LPIAT functioning as a determinant of PI molecular species.In this meeting, we will present our recent data on both mboa-7 knockout worms and knockout mice.

Loer CM et al. (1991) International C. elegans Meeting "A candidate gene for a serotonin synthetic enzyme in C. elegans."

Loer CM, Kenyon CJ

[International C. elegans Meeting, 1991]

The biogenic amine neurotransmitters serotonin and dopamine are present in a number of identified neurons in C. elegans. These transmitters are synthesized (in mammals) by two enzymatic steps from tryptophan and tyrosine, respectively, catalyzed by specific aromatic amino acid (AAA) hydroxylases and a decarboxylase. We have cloned a gene encoding a C. elegans AAA hydroxylase by amplification of genomic DNA with degenerate primers. We have isolated a genomic clone and cDNAs. Preliminary sequence data suggest that the gene encodes a protein of 440 amino acids that falls in the phenylalanine/tryptophan class of hydroxylases. In a comparison of 307 C-terminal amino acids, the encoded protein shared identity with 200 aa of a Drosophila Phe/Trp hydroxylase (unpublished sequence courtesy of Wendi Neckameyer) , 195 aa with Rat PheH, and 178 aa with Rat TrpH. Analysis of genomic DNA shows that the gene also shares some conserved intron sites with the AAA hydroxylase gene family. To see whether this is a good candidate for an enzyme required for serotonin synthesis, we are trying to determine where the gene is expressed. With this and other markers of specific neuronal differentiation, we hope to find genes that determine what neurotransmitter a neuron will use.

Hyeon-Cheol Lee et al. (2008) ""C. elegans mboa-7, a Member of the MBOAT Family, is Required for Selective Incorporation of Polyunsaturated Fatty Acids into Phosphatidylinositol""

Hyeon-Cheol Lee, Hiroyuki Arai, Rieko Imae, Shinichiro Shirae, Shinji Matsuda, Takao Inoue, Nozomu Kono

[2008]

"Phosphatidylinositol (PI) is a component of membrane phospholipids and functions both as a signaling molecule and as a compartment-specific localization signal in the form of polyphosphoinositides. Arachidonic acid (AA) is the predominant fatty acid in the sn-2 position of PI in mammals. LysoPI acyltransferase (LPIAT) is thought to catalyze formation of AA-containing PI; however, the gene that encodes this enzyme has not yet been identified. In this study, we established a screening system to identify genes required for utilization of exogenous polyunsaturated fatty acids (PUFAs) in C. elegans. In C. elegans, eicosapentaenoic acid (EPA) instead of AA is the predominant fatty acid in PI. We showed that an uncharacterized gene, which we named mboa-7, is required for incorporation of PUFAs into PI. Incorporation of exogenous PUFA into PI of the living worms and LPIAT activity in the microsomes were greatly reduced in mboa-7 mutants. Furthermore, the membrane fractions of transgenic worms expressing recombinant MBOA-7 and its human homolog exhibited remarkably increased LPIAT activity. mboa-7 encodes a member of the membrane-bound O-acyltransferase family, suggesting that mboa-7 is LPIAT. Finally, mboa-7 mutants had significantly lower EPA levels in PI and exhibited larval arrest and egg-laying defects."

Glenn E White (2005) International Worm Meeting "Using C. elegans to Determine the Evolutionary Relationship Between Paramyosin and the Conventional Myosin II Tail"

Glenn E White

[International Worm Meeting, 2005]

The coiled coil protein paramyosin is a major component of invertebrate muscle and is homologous to the myosin II tail. Among others, both WormBase and NCBIs Conserved Domain websites characterize paramyosin as a pfam01576.11 myosin tail motif. Because these are websites, this information is included without comment. Amino acid (AA) sequence analysis and molecular phylogenetics were used to examine the possible evolutionary relationship between the two. AA sequence comparisons between C. elegans paramyosin and the last 872 AAs of the myosin A tail from C. elegans revealed that there is 38.4% AA identity (e-159) between the two over 811 AAs. When comparing paramyosin to the first 872 AAs of the myosin A tail, the % AA identity drops to 35.6% (e-107) over 592 AAs. These Expect scores demonstrate why paramyosin was classified as a myosin tail. % AA identity drops when paramyosin is compared to mouse smooth muscle myosin (27.5%, e-104) and to human non-muscle myosin IIC tails(26.6%, e-92) but the Expect scores are consistent with true homology. An unrooted molecular phylogeny for paramyosin was generated first for 25 paramyosins from 24 organisms. The paramyosins sorted as 1) mollusks, 2) flying insects, 3) ticks, 4) mites, 5) tapeworms, 6) flukes, and 7) nematodes. To test the hypothesis that paramyosin and the myosin II tail are evolutionarily related, a molecular phylogeny using myosin II tail AA sequences from several organisms representing all classes of myosin IIs and paramyosin (unc-15 from C. elegans) was prepared. Designating paramyosin as the outgroup results in a single duplication event accounting for the rise of the striated and non-/smooth muscle lineages of all myosin II tails. All paramyosins produce the same result. Conversely, any myosin II tail sequence can root a paramyosin tree. A single duplication of that myosin II tail gives rise to one lineage which includes tapeworms, flukes and mollusks and another lineage which includes nematodes, flying insects, ticks, and mites. These results suggest that there is a strong evolutionary relationship between paramyosin and the myosin II tail, regardless of which myosin it is compared to. When another coiled coil protein such as tropomyosin roots a tree of myosin II tails, additional duplication events are introduced, implying that the paramyosin results are not simply because it is a coiled coil protein. The results indicate that an argument for a strong evolutionary relationship between the two proteins can be made, but cannot distinguish between whether a myosin II gene was split to form paramyosin in invertebrates, or that an ancient paramyosin combined with another molecule (a myosin I perhaps?) to create an ancient myosin II.