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First published online 24 February 2009
doi: 10.1242/jcs.042754

Journal of Cell Science 122, 822-833 (2009)
Published by The Company of Biologists 2009
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Expression of ceramide glucosyltransferases, which are essential for glycosphingolipid synthesis, is only required in a small subset of C. elegans cells

Esther Marza1, Karina T. Simonsen2, Nils J. Færgeman2 and Giovanni M. Lesa1,3,*

1 MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK
3 Department of Cell and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK
2 University of Southern Denmark, Department of Biochemistry and Molecular Biology, Campusvej 55, 5230 Odense, Denmark


Figure 1
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  		Fig. 1. Simplified GSL synthetic pathway. Glucosylceramide (GlcCer) is the precursor of all C. elegans glycosphingolipids. Cer, ceramide; CGT, ceramide glucosyl transferase.

 

Figure 2
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  		Fig. 2. cgt-1, cgt-2 and cgt-3 encode ceramide glucosyltransferases. (A) ClustalX alignment of CGT-1, CGT-2 and CGT-3 proteins with human CGT. Single-letter abbreviations for amino acid residues are used. Identical and similar amino acids are identified by dark blue and orange shading, respectively. The putative transmembrane domain is indicated with a red line. The asterisk indicates a conserved histidine in the UDP-glucose binding pocket essential for optimal enzymatic activity. Green dots indicate amino acids essential for enzymatic activity in vitro (Marks et al., 2001Go). (B) Genomic organization and deletions of the cgt genes. cgt-3 is located on chromosome II, cgt-1 on chromosome V and cgt-2 on chromosome X. The coding regions are boxed and non-coding regions are shown as lines. Numbers at the top and bottom of each gene structure indicate nucleotide and amino acid sequences, respectively. The lines below each gene indicate deletions and their breakpoints. The asterisk and green dots are as in A. (C) Diagram of the comparison of identity among C. elegans and human CGT proteins. Percent identity at amino acid sequence level is shown.

 

Figure 3
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  		Fig. 3. Analysis of cgt-2(tm1097) and cgt-2(tm1192) mRNA. Gel showing the RT-PCR products of total mRNA extracted from wild-type, cgt-2(tm1097) or cgt-2(tm1192) animals with primers GL599 and GL600 (supplementary material Table S1). Both tm1097 and tm1192 mRNAs produce a major band and a minor band (triangles). Below is a schematic with the splicing products corresponding to the mutant bands. Boxes represent exons (numbered in white). Numbers at the top and at the bottom of cgt-2 gene structure indicate nucleotide and amino acid sequence, respectively. The asterisk indicates the conserved histidine in the UDP-glucose binding pocket. Green dots indicate amino acids essential for CGT enzymatic activity (Marks et al., 2001Go).

 

Figure 4
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  		Fig. 4. Glucosylceramide levels in CGT mutant animals. (A) Thin-layer-chromatography analysis of polar lipids extracted from various CGT mutants (lanes 1-4 and 6) and from wild-type animals at different developmental stages (lanes 7 and 8). The origin is always at the bottom. The migration area of glucosylceramides (GlcCer) and ceramides (Cer) are indicated. A band similar to that observed in bacteria (asterisk) was present in other lanes. Since its intensity was proportional to the amount of bacteria in the sample, it is unlikely that wild-type animals produce lipids overlapping with this band. (B) Histogram showing the average relative intensity of the GlcCer bands, which include GSLs, from three separate experiments. GlcCer levels were not affected in cgt single mutants (lanes 1, 2 and 4), but were severely decreased when all CGT genes were knocked out or knocked down (lane 3). GSL levels from arrested CGT animals (lanes 3 and 6) were compared with those of starved wild-type L1 animals (lane 8). GSL levels from mixed-stage mutant populations (lanes 1, 2 and 4) were compared with those of mixed stage wild-type populations (lane 7). GlcCer levels in starved wild-type L1 animals were similar (89.8±6.1%; not shown) to those of wild-type animals (100%). The bands quantified (arrowhead in A) were those above the major bacterial band. Values are expressed as % wild-type ± s.e.m.

 

Figure 5
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  		Fig. 5. Essentially eliminating CGT function results in arrest at the first larval stage (L1). (A) Wild-type animals or single CGT mutants reach adulthood within ~3.5 days at 20°C. The inset shows a wild-type L1 animal. Scale bar: 100 µm. (B) cgt-3(RNAi);cgt-1(qa1809) mutants appear normal as soon as they hatch (left inset), do not develop beyond the L1 stage and die after a few days (right inset). Scale bar: 100 µm. (C) Electron micrograph of a transverse section of an arrested cgt-3(RNAi);cgt-1(qa1809) mutant worm. Arrows indicate cuticular ridges (alae) present at the L1 stage but not at other larval stages. The cgt-3;cgt-1 double mutant worm shown has therefore arrested at the L1 stage. Scale bar: 1 µm. (D,E,F) Worms carrying an integrated hlh-8::gfp reporter gene, which directs GFP expression in the M-cell lineage. M-cell number reflects the developmental stage of L1 larvae. Worms hatch with one M cell (D); subsequent divisions produce 2, 4, 8 and, by the end of L1, 16 M cells (E). Scale bars: 10 µm Approximately 45% of cgt-3(RNAi);cgt-1(qa1809);cgt-2(tm1192) worms arrested at the one-M-cell stage, 39% at the two-M-cell stage, 5% at the eight-M-cell stage and 11% at the 16-M-cell stage (F). ***P<0.0001.

 

Figure 6
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  		Fig. 6. Expression pattern of cgt-1 and cgt-3 in L1 larvae. CGT-1::GFP (A-C,G-I,M-O) and CGT-3::GFP (D-F,J-L,P-R) show both a strong signal in pharyngeal muscles (diamond), PIV (triangle), intestinal cells (asterisk), and IRV and RGCs (both indicated by a square). Close views of the PIV (G-L) and the IRV and RGCs (M-R) are shown. The pharynx is marked with a red dashed line; the PIV with a green dashed line. For expression in adults, see supplementary material Fig. S1. Scale bar: 10 µm.

 

Figure 7
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  		Fig. 7. GSL levels are close to normal in CGT triple mutants expressing cgt-1 under the control of the F54D5.1 promoter. (A-C) F54D5.1p::gfp is expressed in a limited number of cells: the PIV, the IRV, the RGCs and often the most anterior and posterior intestinal cells. Scale bar: 10 µm. (D) Thin-layer-chromatography analysis of polar lipids extracted from CGT triple mutants (lane 1), triple mutants expressing F54D5.1p::cgt-1 (lane 2) and wild-type animals (lane 3). Alleles: cgt-1(qa1809) (lane 1), cgt-1(tm1027) (lane 2), cgt-2(tm1192), cgt-3(tm504).

 

Figure 8
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  		Fig. 8. CGT mutants are unable to ingest food properly and starve. cgt-1(qa1809);cgt-2(tm1192) L1 larvae (control, A-C) or arrested cgt-1(qa1809);cgt-2(tm1192) treated with cgt-3 RNAi (D-I) animals were exposed to fluorescent beads mixed with food for 2 hours. Beads were detected in the intestinal tract (dashed red line in C) of most control animals, whereas most cgt-3;cgt-1;cgt-2 worms had beads stuck just before (D) or just after (G) the PIV (yellow dashed line in F and I). (J) Quantification of fluorescent beads in the digestive tract. Mean ± s.e.m.; ***P<0.0001. (K-N) DAF-16::GFP localization in L1 larvae. DAF-16::GFP localizes in the cytoplasm under normal conditions and in the nucleus under starvation/stress conditions (Henderson and Johnson, 2001Go). In wild-type (K) and cgt-1(qa1809);cgt-2(tm1192) (M) L1 animals, DAF-16::GFP is detected in the cytoplasm, while it is mainly nuclear in cgt-3(RNAi);cgt-1(qa1809);cgt-2(tm1192) (N), and in daf-2(RNAi) animals (L). DAF-2 normally inhibits DAF-16 nuclear localization. Scale bars: 10 µm.

 

Figure 9
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  		Fig. 9. CGT triple mutants show abnormal morphology of the intestinal and PIV cells. (A-C) Wild-type L1 animals expressing F54D5.1p::gfp in the PIV and in the IRV. The intestinal lumen appears thin and continuous. (D-L) cgt-3;cgt-1;cgt-2 L1-arrested larvae expressing F54D5.1p::gfp. (D-F) CGT triple mutants arrested for 1 day showed morphologically normal PIV cells (surrounded by a green dashed line, E). However, the anterior intestinal cells appeared swollen and vacuolated (white arrow, E), and the intestinal lumen was enlarged. A red dashed line surrounds the pharynx (E). (G-L) After 3 days of L1 arrest, CGT triple mutants showed more severe defects. The PIV cells lost most of their fluorescence (spot in G), suggesting that they were dying, or became swollen (spots, K) with diffuse GFP staining (J-L). The anterior intestine appeared disorganized (arrow, H) and the intestinal tract was discontinuous in some places (arrowhead, H). Alleles: cgt-1(qa1809), cgt-2(tm1192), cgt-3(RNAi). Scale bars: 10 µm.

 

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© The Company of Biologists Ltd 2009