elegans lipid extracts

Synchronized L1 larvae of wild type Bristol N2, hyl-2(gnv1), 2(tm231), hyl-1(ok976) and hyl-1(gk203) were obtained from hypochlorite-treated adults and were grown on NGM plates for three days at 20°C and harvested using double-distilled water. Standards DMPC (5 µg), DMPE (20 µg), DLPE (20 µg), DLPS (20 µg), diC8-PI (4 µg), C17 cer (3 µg), C12 SM(3 µg) and C8GC(3 µg) were added to a pellet of 100mg of nematodes. Animals were resuspended to 1ml with double-distilled water and disrupted in a beadbeater (Mini-Beadbeater-96, Biospec) with glass beads of 0.5mm (ref PEC-11079105, Milian). Worm homogenate were mixed at room temperature with 3.2 ml of CHCl3:CH3OH 1:2 (v/v) and gently centrifuged for 15 minutes (200g). 1ml of CHCl3 and 1ml of CH3OH were added to the cleared supernatant and gently centrifuged for 10 minutes at 4°C. The collected organic phase was washed once with double distilled water (ddwater), dried with nitrogen (N2 4.5, with ddwater, dried with nitrogen (N2 4.5, Pangas) and kept at -20°C. All extractions steps were performed in acid washed glassware.

Lipid profiling by Mass Spectrometry

Preliminary experiments were performed to analyze the profiles of sphingomyelin (SM), ceramide (Cer) and phosphatidylcholine (PC) species in N2, 1(ok976), hyl-1(gk203), hyl-2(gnv1) and hyl-2(tm2031) worms. Lipids were introduced into a Varian 320MS triple quadrupole in LC mode by direct infusion in methanol:chloroform:H2O (7:2:1, containing 2 mM ammonium acetate). SM and PC were detected using precursor ions scans in the positive ion mode detecting the

choline headgroup (m/z=184). SM was differentiated from PC by base hydrolysis of the samples, removing the base-sensitive PC. This also greatly reduced ion suppression of SM by PC. The identity of SM species was confirmed by Q3 scan to detect the C17 isobranched sphingosine base (m/z=250.2) after water loss. Cer was detected by precursor ion scans using the C17 isobranched sphingosine base. The optimized capillary and collision energies were 130V, 22V for PC, 97 V, 22 V for SM, and 50 V, 22 V for Cer. To quantify lipid species multiple ion monitoring (MRM) was used for each species. MRMs were collected from at least two independent samples with at least duplicate measurements. The amounts of different species were calculated relative to the internal standards from Avanti Polar Lipids (C17 ceramide, C12 sphingomyelin, DMPC). Therefore, the amounts found should not be considered to be absolutely precise because the C. elegans lipid species, which are not available in pure form, are not identical to the standards. On the other hand, the relative quantities of the individual lipid species in the different strains should be accurate. Standard deviations were calculated and error bars in the figures represent plus and minus one standard deviation.

In Vitro assay of dihydroceramide synthase

Synchronized young adults were harvested with M9, resuspended in 3ml Tris-EDTA (TE, pH7.5) + protease inhibitors (Complete Protease Inhibitor, Roche) and disrupted in a Mini-beadbeater as described previously. Worm debris was pelleted by 10 min centrifugation (200g). Supernatants were centrifuged for 30 min (100,000g). The pellets containing light membrane fraction were resuspended in 200µl TE (pH7.5) + protease inhibitors. Protein concentrations were determined by Bradford (BioRad Protein Assay) according to manufacturer’s protocol. Samples were quickly frozen with liquid nitrogen and kept at -20°C. LMF were assayed as previously described (7) using ³H-sphinganine and acyl-CoAs of different lengths. The reaction products were isolated and dihydroceramide was separated by thin layer chromatography (solvent, CHCl3:CH3OH, 100:7) and exposed to a phosphorimager screen. Membranes from hyl-2 mutants (hyl-2(gnv1) and hyl-2(tm2031)) synthesized less C22 dihydroceramide, while hyl-1 mutants (hyl-1(gk203) and hyl-1(ok976)) synthesized less C26 dihydroceramide. All membranes synthesized C24 dihydroceramide equally.

Supporting References

S1. S. Brenner, Genetics 77, 71 (May, 1974).

S2. K. G. Miller et al., Proc Natl Acad Sci U S A 93, 12593 (Oct 29, 1996).

S3. P. Zipperlen et al., Genome Biol 6, R19 (2005).

S4. C. C. Mello, J. M. Kramer, D. Stinchcomb, V. Ambros, Embo J 10, 3959 (Dec, 1991).

S5. D. Mumberg, R. Muller, M. Funk, Gene 156, 119 (Apr 14, 1995).

S6. B. Zanolari et al., Embo J 19, 2824 (Jun 15, 2000).

S7. N. Kageyama-Yahara, H. Riezman, Biochem J 398, 585 (Sep 15, 2006).

Supporting Tables and Figure Legends Table S1

List of the mutants tested for their sensitivity to anoxia. Animals were synchronized and tested 72 h post L1-stage and exposed to 48 anoxia. (-- not sensitive, +++ highly sensitive).

Table S2

Identification of positive ions seen by ESI-MS. The possible identities of ceramides (Cer), sphingomyelins (SM) and phosphatidylcholines (PC) species seen by MS are shown. The nomenclature is (# carbons in the acyl chain(s): # desaturations: # hydroxylations). In several cases it was not possible to differentiate between nearly isobaric species with the resolution permitted by our instrument. However, desaturations in the acyl chains of Cer and SM have not been reported for C. elegans, so in each case where there are two possibilities for Cer and SM the most likely one is the fully saturated acyl chain. The possibilities shown for PC species are not exhaustive.

Fig. S1: Morphological alterations in hyl-2(gnv1) animals post-anoxia.

A-D: Contrast phase (A,C) and propidium iodide (PI) staining (B, D) of worms that survived 16 h anoxia. E-J. Contrast phase (E, G, I) and PI staining (F, H, J) of worms that did not survived anoxia. Note that live worms display a faint staining in the lumen of the anterior part of the pharynx that may represent bacteria (white arrow).

Dead worms can show vacuoles surrounding the pharynx (E, black arrow), PI positive nuclei scattered throughout the cell body (white arrowhead) or massive necrosis.

Fig. S2: Ageing of hyl-1 and hyl-2 mutants

Animals were grown in the presence of 5-fluoro-uracil for two days to prevent growth of progeny. Animals were scored every day and considered as dead when they did not respond to gentle touch with a platinum wire. Life span is defined as the day animals were at the L1 larval stage (time t = 0) until the day they were scored as dead. Results are mean of three independent experiments.

Fig. S3. hyl-2(gnv1) and hyl-2(tm2031) animals are sensitive to heat shock.

A. Survival of hyl-1 and hyl-2 mutants after 3 h incubation at 36°C. Results are mean

± SD (n=3). B. Survival of hyl-2(gnv1) mutants complemented with the transgene GFP::hyl-2 under the control of hyl-2 promoter. Survival was examined after 3 h incubation at 36°C. Results are mean ± SD (n=4).

Fig. S4: Schematic representation of the various strains and constructs used to generate transgenic worms.

Fig. S5. In vitro assay of dihydroceramide synthase. Light membrane fractions were prepared from N2, hyl-2(gnv1), hyl-2(tm2031), hyl-1(ok976) and hyl-1(gk203) nematodes, which were assayed as previously (7) using ³H-sphinganine and acyl-CoAs of different lengths (Avanti Polar Lipids). The reaction products were isolated and dihydroceramide was separated by thin layer chromatography (solvent CHCl3:CH3OH 100:7) and exposed to a phosphorimager screen. Membranes from hyl-2 mutants synthesized less C22 dihydroceramide, while hyl-1 mutants synthesized less C26 dihydroceramide. All membranes synthesized C24 dihydroceramide equally.

These results agree well with the mass spectrometry results.

Fig. S6. Estimate of the total amount of Cer, SM and PC in wild type and mutant worms.

All species of Cer, SM, and PC quantified by ESI-MS were added together to obtain an estimate of the total amount these lipid classes in wild type and mutant worms.

Results are mean ± SD (n=4).