Chapter II Materials and Methods

CHAPTER II

MATERIALS AND METHODS

Strains and strain maintenance:

C. elegans cultures were grown on solid medium as described by Brenner (Brenner, 1974), modified by the addition of streptomycin and mycostatin to reduce contamination, and the use of the streptomycin-resistant bacterial strain OP50/1 (Johnson et al., 1988). The nlg-1(ok259) mutant was provided by the C. elegans Gene Knockout Consortium. The pha-1(e2123) mutant was provided by the Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN). The nlg-1(tm474) mutant was obtained from Shohei Mitani (Tokyo Women's Medical College, Tokyo, Japan).

Sequencing of nlg-1 mutants:

nlg-1 deletion mutations were analyzed by amplification of specific nlg-1 genomic regions from mutant animals (Barstead, 1989), followed by sequencing of the purified PCR product with nested primers. DNA sequencing was performed at the OMRF DNA Sequencing Core Facility, using oligonucleotide primers obtained from IDT (Coralville, IA).

Analysis of nlg-1 transcripts and alternative splicing:

RNA was isolated from wild-type C. elegans using standard procedures. Alternative splice forms were analyzed using RT-PCR with exon-specific primers, followed by DNA sequencing with internal primers. The 5’ ends of mRNAs were analyzed using the Invitrogen 5’ RACE System for Rapid Amplification of cDNA Ends, Version 2.0 using oligonucleotide primers.

The cDNA clone yk497a9 was obtained from Yuji Kohara (National Institute of Genetics, Mishima, Japan) and the insert was fully sequenced (GenBank accession FJ825295); it includes 51 nucleotides of 5'- UTR (out of a predicted 71 nucleotides), 2526 nucleotides of coding sequence (including both exons 13 and 14; see Figure 8 and Figure 9), 276 nucleotides of 3'-UTR, and a 19-nucleotide polyA sequence.

Reporter constructs and generation of fusion proteins:

Plasmids containing the CFP, YFP or GFP coding sequences were derived from the pPD95.67, pPD132.12 or pPD133.48 plasmids as per Miller et al. (Miller et al., 1999a). A plasmid containing a modified mCherry ("wormCherry") gene, described by McNally et al. (McNally et al., 2006) and Green et al. (Green et al., 2008), was a generous gift from Anjon Audhya and Karen Oegema (Ludwig Institute for Cancer Research, La Jolla, CA). Some reporter constructs were generated using a PCR fusion approach with overlapping PCR primers (Splicing by Overlapping Extension PCR or SOEing PCR) as described by Horton et al. (Horton et al., 1989) and Hobert et al. (Hobert, 2002). The neuroligin transcriptional fusion (FRM77) (Figure 8 and Figure 9) was generated by PCR-amplifying the 5’-end of the nlg-1 gene (from 3563 base pairs upstream of the SL1 trans-splice site through the first 45 base pairs of exon 3), and fusing this product in-frame to the YFP coding sequence followed by the unc-54 3’-UTR. The neuroligin functional fusion protein (FRM253) (Figure 8 and Figure 9) had the YFP inserted after amino acid E661 (Figure 7) (using the protein sequence derived from the yk497a9 cDNA; GenBank accession ACO52513). FRM253 was generated by amplifying a PCR product containing the same upstream region used to make FRM77, but extending downstream to amino acid 661 at the end of exon 11. This amplification product was fused in-frame to the YFP coding sequence, the amino acids 659-661 were repeated, followed by nlg-1 exons 12-16 and the nlg-1 3’-UTR, which were derived from the yk497a9 cDNA. The structures of these reporters are shown in Figure 8 and Figure 9. We used the PCR product made from pPD132.12 (described above) as a “megaprimer” in a Quickchange™ reaction to insert YFP into yk497a9, generating the plasmid RM#980p, which was used in lieu of a three-way SOE (Splicing by Overlapping Extension PCR) for some experiments. Briefly, the “megaprimer” was used to do 25 rounds of thermo-cycling using Pfu polymerase and yk497a9 cDNA as a template; the resulting product was digested with DpnI and then transformed into DH5α cells.

Modification of mammalian NLGN cDNAs:

To generate the human NLGN4 and rat Nlgn1 transgenic animals, we started with the mammalian cDNAs (gift from T. Südhof, Stanford U.) and used Quickchange™ to replace the signal sequence and the 3’-UTR with the C. elegans NLG-1 signal sequence and the nlg-1 3’-UTR, respectively. The “megaprimers” for these Quickchange reactions were generated from yk497a9 in the plasmid RM#980p. The resulting clones were sequenced as described above. Fragments with the nlg-1 promoter driving the modified mammalian cDNAs were generated in the same manner as described for reporter constructs.

Generation of ASD mutant YFP fusion proteins and deletion construct YFP fusion proteins:

Primers with the mutation (either primers with mutations homologous to the ASD associated mutation or primers that loop out the sequence to be deleted) flanked by homologous sequence were used to perform 25 rounds of thermo-cycling using Pfu polymerase and RM#980p as a template; the resulting product was digested with DpnI and then transformed into DH5α cells. The resulting clones were sequenced as described above. Fragments with the nlg-1 promoter driving the modified cDNAs were generated in the same manner as described for reporter constructs.

Transgenic methods:

Transgenic nematodes were obtained by microinjection of DNA (plasmids and/or PCR products), essentially as described by Mello et al. (Mello et al., 1991). Transformation markers included the pBX plasmid (Heinke and Ralf Schnabel, Max-Plank-Institute fur Biochemie), which was reported by Granato et al. (Granato et al., 1994) to rescue the temperature sensitive lethality of pha-1(e2123) mutants. For experiments utilizing the pBX plasmid, we constructed appropriate recipient strains for transformation containing the pha-1(e2123) mutation. For some DNA injections, we used a modified unc-122 promoter to drive GFP expression in coelomocytes (Loria et al., 2004). Extrachromosomal arrays were integrated randomly into chromosomes by gamma irradiation essentially as described by Schade (Schade et al., 2005).

Cell identification methods and reagents:

nlg-1-expressing cells were identified on the basis of position and morphology as defined by the Worm Atlas by Altun and Hall (WormAtlas, 2002-2006) and also through the use of specific transgenic reporters (Chalfie et al., 1994, Boulin et al.) for colocalization and/or to provide cellular landmarks. The cells identified as nlg-1-positive were found to express the Pnlg-1::YFP reporter (FRM77) in 100% of animals observed, with at least 10 animals examined for each genotype. The promoters used for these studies included unc-25 for GABAergic neurons (Eastman et al., 1999, Boulin et al.), unc-17 for cholinergic neurons (Duerr et al., 2008, Alfonso et al., 1993), eat-4 for a subset of glutamatergic neurons (Lee et al., 1999b), dat-1 for dopaminergic neurons (Sulston et al., 1975, Nass et al., 2002), tph-1 for serotonergic neurons (Sze et al., 2000), acr-5 for DB and VB motor neurons (Winnier et al., 1999), ttx-3 for AIY interneurons (Altun-Gultekin et al., 2001), gcy-8 for AFD sensory neurons (Yu et al., 1997), glr-3 for RIA interneurons (Brockie et al., 2001a), odr-2(2b) for AIB, AIZ, ASG, AVG, IL2, PVP, RIF, RIV and SIAV neurons (Chou et al., 2001), and nmr-1 for AVA, AVD, AVE, RIM, AVG and PVC interneurons (Brockie et al., 2001b). Dye-filling of ciliated sensory neurons with DiI as described by Hedgecock (Hedgecock et al., 1985) provided additional cellular landmarks in the head.

Behavioral assays:

All behavioral measurements were performed at ~22°C unless otherwise stated. Statistical significance tests used the Mann-Whitney U test with a criterion p value of p<.05. Swimming rates were measured on hermaphrodites raised at 20°C as described previously (Miller et al., 1999a).

Spontaneous reversal assays:

To score reversal behavior, worms grown at 20°C were transferred to room temperature chemotaxis plates (Hart (ed.)) without bacteria. They were allowed to equilibrate for two min. and then were scored visually for either forward and reverse movements for ten min. using the Etho 1.2.2 program (provided by Dr. James H. Thomas, Genome Sciences, University of Washington). Only changes in direction were scored, and therefore pauses were disregarded.

Thermotaxis assays:

Thermotaxis assays were performed by establishing a thermal gradient on a 100mm chemotaxis plate without food by placing a vial of frozen acetic acid (16.7°C) beneath the center of the plate, which was placed into a 25°C incubator (Hedgecock and Russell, 1975). ~50 worms were transferred to the temperature gradient plate and allowed to move freely for 30 min. Worms were then counted on an overlay of concentric circles demarking eight equal areas. To assign a numeric value for determining degree of rescue, the percentage of animals in 19, 20 and 21°C were summed (animals who are thermotracking).

Chemotaxis assays:

Chemotaxis assays were performed on chemotaxis plates (Hart (ed.)) without food. A chemo-attractant (2 µl of 0.1% diacetyl in ethanol) or repellant (2 µl of 0.1% octanol in ethanol) was placed on one side of the plate (A) and 2 µl of ethanol was placed on the opposing side of the plate (B). ~50 worms were placed in the middle of the plate and were allowed to move freely for 20 min., and then counted. A chemotaxis index (C.I.) was calculated using the formula C.I.=A-B/A+B. Only worms within 2 cm of the test spots were scored, and therefore worms in neutral areas were disregarded.

Approach avoidance:

To test attraction and avoidance simultaneously, a chemo-attractant (2 µl of 0.1% diacetyl) was placed on one side (A) of the chemotaxis plates (Hart (ed.)) without food, ~50 worms were placed on the opposing side (B) of the plate and a barrier of cupric acetate (50 µl of .5mM Cu(CH₃COO)₂) was placed in a line across the middle of the plate. The worms were allowed to move freely on the plate, and the worms that crossed the barrier were scored. An Approach-Avoidance (A-A) ratio was calculated using the formula A-A ratio=A/A+B.

Immunofluorescence Staining of Nematodes

Nematodes were stained using a freeze-fracture procedure adapted from Duerr et al. (Duerr et al., 1999), with major modifications by Kiely Grundahl (Mullen et al., 2006). Nematodes were freeze fractured (with no subbing agent) and then washed from slides in ice-cold methanol. Nematodes were pelleted in 50-ml conical tubes, transferred to microfuge tubes, and then pelleted again. Nematodes were then fixed in acetone for 2 min, washed in phosphate-buffered saline (PBS), and then blocked with 10% donkey serum in antibody buffer (0.5% Triton X-100, 1 mM EDTA, and 0.1% bovine serum albumin in PBS with 0.05% sodium azide) (Duerr et al., 1999). Primary anti-GFP (Molecular Probes; Eugene, OR) and anti‑RIM (gift from Dr. Mike Nonet, Washington University) antibody incubations (1:50 –1:100) were done overnight at 4°C. After thorough washing with antibody buffer, nematodes were incubated in secondary antibodies overnight at 4°C. Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). After washing, nematodes were mounted in anti-fade medium (20mM Tris pH 9.0, 0.5% N-propyl gallate, 70% Glycerol with 8 µl DAPI) (Duerr et al., 1999).

Microscopy and Imaging:

Confocal images were collected on a Leica TCS NT confocal microscope. Low-resolution images were collected with a 40x Plan Fluotar 1.0 NA oil immersion objective, at 1024x1024 pixels, with 0.5 μm Z-steps. Some florescence images and DIC micrographs were obtained on the NIKON TE2000-U inverted microscope equipped with filter wheels and CoolSNAP ES digital camera (Roper Scientific, Inc.) using MetaVue 6.1 software (Universal Imaging, West Chester, PA). Images were cropped to size, assembled, and annotated using Adobe Photoshop® Elements. Digital manipulations were limited to resizing (Photoshop® Bicubic) and minor levels adjustments.

Toxicity studies:

Paraquat sensitivity assays:

Survival was assessed by placing 20 young adult worms on NGM-L plates containing paraquat (1.8 mM) and a bacterial lawn of food, and scoring worms as living or dead every 24 hours.

Heavy metal toxicity:

Survival was assessed by placing 20 young adult worms on NGM-L plates containing a test heavy metal and a bacterial lawn of food, and scoring worms as living or dead every 24 hours. Heavy metals included mercury (as thimerosal (either 6 or 12 µM C₉H₉HgNaO₂S) or mercury salt (0.7 µM HgCl₂)), cadmium (8.0 mM CdCl₂) and copper (0.7 mM Cu(CH₃COO)₂).

Aldicarb assays

Neurotransmission was assessed using aldicarb (a cholinesterase inhibitor which prevents the breakdown of acetylcholine in the synapse). Acute sensitivity was assessed by plating 20 young adult worms onto NGM-L plates with food (OP50 E. coli) containing 2mM aldicarb and then scoring worms for paralysis every 20 minutes for three hours (Schade, 2005 ). Chronic aldicarb survival was assessed by plating 5 young adult worms onto NGM-L plates with food (OP50 E. coli) containing 0.5mM aldicarb and then scoring the number of progeny after three days (Miller et al., 1999b).

Oxidative damage ELISA:

Biochemical assays were performed by putting several hundred young adult worms on NGM-L plates with or without 1.8 mM paraquat and a bacterial lawn of food for 2 days (1 day for mev-1 mutant), harvesting them in chilled M9 buffer and freeze powdering them in liquid nitrogen. Proteins were extracted in “lysis buffer” (20 mM 2-(N-Morpholino)-ethanesulfonate (MES) buffer pH 5.5 containing 0.1% triton X-100, complete protease inhibitor cocktail (1 tablet/50ml) (Roche Diagnostics) and 100 μM butylated hydroxytoluene (BHT)) and were subjected to two 15 second pulses of sonication with a Sonic Dismembrator model 100 (Fisher Scientific). The protein concentrations of the samples, the oxidized BSA standard and the reduced BSA standard were measured by the Micro BCA spectro-photometric assay (Pierce), and then the protein concentration was adjusted with “lysis buffer” to 5 µg/ml. Duplicate 200 µl of the diluted standards, samples (10 µg protein in 1 ml PBS), and PBS without protein (blank) were added into the wells. DNPH solution (200 µl) was added, incubated for 45 min. at room temperature in the dark and then neutralized with 50µl 0.1N NaOH. The samples were transferred to a new plate, incubated overnight at 4ºC and then was washed three times with 300 µl PBS:ethanol (1:1, v/v) and three times with 300 µl PBST (PBS, 0.1% Tween 20). Blocking solution (270 µl) was added and incubated for 1.5 h at room temperature and then washed five times with 300 µl PBST. Anti-DNPH (200 µl) was added, incubated for 1 h at 37ºC and then washed five times with 300 µl PBST. Secondary antibody (200 µl) was added, incubated for 1 h at 37ºC and then washed five times with 300 µl PBST. 200 µl Super Aqua Blue ELISA Substrate (eBioscience) was added, and the absorbance was read at 405 nm.

For reduced BSA standard, one gram of BSA was dissolved in 100 ml PBS. Then 0.1 g of solid NaBH₄ was added and incubated for 30 min at room temperature, neutralized with HCl (2 N) that was added slowly under the hood, followed by overnight dialysis against PBS at 4ºC (with two buffer changes) dispensed in aliquots, and then stored at -80ºC. Oxidized BSA standard was prepared by using 50mg of BSA dissolved in 1 ml PBS, 20 µl EDTA (100 mM), and 100 µM Fe³⁺/25mM ascorbate was added, incubated for 1.5 h in 37ºC , followed by overnight dialysis against PBS at 4ºC (with two buffer changes), dispensed in aliquots, and then stored at -80ºC . For the DNPH solution (0.05 mM, pH 6.2, freshly prepared), 1.2 mg DNPH was dissolved in 333 µl phosphoric acid (85%) and added to 40 ml dH₂O. The pH was adjusted to 6.2 by adding drops of 10 M NaOH, and the final volume was adjusted to 50 ml. For the blocking solution (5%), 2.5 g skimmed milk was dissolved in 50 ml PBS. For the primary antibody (freshly prepared), 25 µl of anti-DNPH antibody and 20 µl Tween-20 were added to 10 ml blocking solution. For the secondary antibody (freshly prepared), 25 µl of anti-rabbit antibody HRP-linked IgG and 20 µl Tween-20 were added to 10 ml blocking solution.

Analysis of tissue aging:

Lifespan and health span were analyzed in two ways. Eggs were transferred to seeded NGM-L plates, allowed to hatch, and adults were transferred away from their progeny. Survival of three groups of 25 animals was assessed for each genotype by scoring worms as living or dead every 24 hours (death was defined as the absence of pumping and movement when prodded). Animals were also scored for spontaneous movement.

Nomarski images for 12 animals of each genotype for each day from age 8 days post hatch to age 12 days post hatch were collected on a NIKON TE2000-U inverted microscope equipped with filter wheels and CoolSNAP ES digital camera (Roper Scientific, Inc.) using MetaVue 6.1 software (Universal Imaging, West Chester, PA). Images were collected with a 40x objective with the grinder of the pharynx in sharp focus. Vacuolar lesions between the posterior end of the posterior bulb of the pharynx and the tip of the nose were counted.

BHT antioxidant treatment:

To assess the effect of antioxidant on survival (for both normal lifespan and paraquat survival) 200μl of 100μM BHT (dissolved in warm 95% Ethanol) was spread onto NGM-L plates seeded with OP50. For lifespan, eggs were transferred to plates, allowed to age, and adults were transferred away from their progeny. Survival of three groups of 25 animals was assessed for each genotype by scoring worms as living or dead every 24 hours (death was defined as the absence of pumping and movement when prodded). For paraquat assays, survival was assessed by placing 20 worms on NGM-L plates containing paraquat (1.8 mM), a bacterial lawn of food and BHT, and scoring worms as living or dead every 24 hours.

dsRNA feeding:

Tubes containing 2 ml of 2×YT media supplemented with 75 μg/ml of ampicillin were inoculated with the host cell + plasmid from the Ahringer library (Source BioScience LifeSciences) and cultured overnight at 37°C. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM, and the culture was incubated at 37°C for 2–4 hours with shaking. The cells were concentrated by centrifugation and then applied directly onto three NGM-L plates (without streptomycin) containing 0.4 mM IPTG for each RNAi experiment. ~10 young adult rrf-3(pk1426); mdIs168 (Pnlg-1::NLG-1::YFP) transgenic worms were put onto each plate. Three plates of worms were grown for each strain of RNAi plasmid. When the worms had grown for several generations and were crowded but not starved, they were washed off with M9 buffer, fixed and stained.

Mutagenesis:

An asynchronous population of worms was harvested from three 60mm plates with M9 buffer and spun down (30 seconds at 150x g) in a 15 ml conical tube. The worms were washed three times with M9 buffer to remove excess bacteria, resuspended in 2 ml M9 buffer and added to 2 ml of 2X EMS (ethane methyl sulfonate) solution (12 μl EMS in 2 ml M9 buffer solution = 60 μM EMS). The tube was mixed well to get the EMS into suspension (giving a final concentration of 30 μM EMS). The tightly capped conical tube was wrapped with parafilm and placed on a rocker in a fumehood for 4 hours at room temperature. The EMS solution was removed by spinning down the worms for 30 seconds, removing the supernatant and washing three times with M9 buffer. The worms were resuspended in 0.5 mL M9 buffer and plated onto seeded 100 mM NGM-L plates. After 24 hr, gravid P₀ adults were harvested and treated with 3–6% sodium hypochlorite (NaClO neutralized with NaOH) to obtain eggs (F₁ embryos), which were allowed to hatch on unseeded 60 mm NGM-L plates, at a density of approximately 100-200 worms per plate.

Mutant Screen:

Five L1 stage F₁ worms per plate were transferred to NGM-L plates seeded with food, and allowed to grow for several days until the cultures were crowded but not starved. The worms were washed off the plates with M9 buffer, and allowed to settle through 10 mL of M9 buffer to wash off any bacteria. The worms were spun down (30 seconds at 150 x g) in a 15 ml conical tube, and transferred to unseeded chemotaxis plates. Octanol avoidance (see chemotaxis assays above) was used as a primary screen. Worms that failed to avoid octanol were then scored for spontaneous reversal (see spontaneous reversal above) as a secondary screen.