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A novel thioredoxin-like protein encoded by the C. elegans dpy-11 gene is required for body and sensory organ morphogenesis

Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

View larger version (98K): [in a new window]   Fig. 1. Body and tail phenotype of (A,G) wild-type worms, and mutants carrying various dpy-11 alleles: (B) e504, (C) s360, (D,I) e224, (H) e431, (J) s261. (E,K) Mutant worm rescued by p72WCP and (F,L) wild-type worm with dpy-11 RNAi. Scale bar: 100 µm (A-F); 20 µm (G-L).

View larger version (71K): [in a new window]   Fig. 2. Cellular features of wild type (A,C,E,G) and dpy-11 (e224) (B,D,F) or dpy-11(RNAi) (H) male tails as visualized by (A,B) Nomarski optics; (C-H) cell-type specific markers to identify: hypodermal cells (C,D; marked by pd11GFP-N), structural cell (E,F; marked by ram-5::gfp) and neuronal processes (G and H; marked by sek-1::gfp). Scale bars: 20 µm.

View larger version (22K): [in a new window]   Fig. 3. The dpy-11 locus and the predicted protein structure. (A) Cosmids and their deletion derivatives used for defining the dpy-11 locus. (B) Genomic organization of dpy-11 and the lesions in mutant alleles. (C) Predicted structure of the DPY-11 protein. The GenBank accession number of the dpy-11 gene is AF250045.

View larger version (98K): [in a new window]   Fig. 4. Expression patterns of dpy-11 and subcellular localization of its product. (C,E,G) Nomarski images of D,F,H, respectively. (A) dpy-11 promoter-driven gfp reporter with a nuclear localization signal in L3 larva. (B) Cytosolic gfp reporter in L4 larva showed no seam cell expression (arrowhead). (C,D) The DPY-11 tagged with GFP at the C-terminus (pd11CTGFP) presented a heterogeneous cytosolic expression pattern in the hypodermis around the vulva. (E,F) The fusion transgene encoding full-length DPY-11 with a GFP tagged between the transmembrane domain and the C-terminus (pd11AGFPC) presented a heterogeneous cytosolic expression pattern in the body hypodermis. (G,H) In gastrulating embryos, cytosolic expression (pd11CTGFP) was detected in unfused hypodermal cells (arrows) with membrane association detected in some dying cells (arrowheads). Scale bars: 100 µm (A,B); 10 µm (C-H).

View larger version (83K): [in a new window]   Fig. 5. Amino acid sequence alignment of DPY-11. (A) Thioredoxin domains in different organisms: C. elegans (Q09433), C. nephridii (X14630), E. coli (K02845), H. sapiens (J04026). (B) DPY-11 homologs in human (AL080080) and fruitfly (AAF47072). The numbers in brackets are GenBank accession numbers.

View larger version (66K): [in a new window]   Fig. 6. Expression and purification of the GST fusion construct. WT represents the GST fusion product of the TRX-domain from amino acids 19-133 of DPY-11. G76E is a similar fusion protein with the glycine at position 76 changed to glutamic acid. GST is the expression of an empty pGEX-2T GST vector, which produced only the GST tag.

View larger version (13K): [in a new window]   Fig. 7. Insulin reduction assay of GST-DPY-11 protein. (A) A time course of the enzymatic activity assayed at a fixed concentration (3 µM) of the tested proteins. (B) A comparison of the catalytic activities of the fusion protein and thioredoxin at different concentrations and a fixed reaction time of 20 minutes. Black squares represent the E. coli recombinant thioredoxin; white triangles represent the GST fusion protein of wild-type TRX domain; black triangles represent the mutant fusion protein with glycine 76 mutated to glutamic acid; white circles represent the GST alone and black circles, the buffer alone.

View larger version (26K): [in a new window]   Fig. 8. A schematic representation of different mutant constructs made for the detection of rescue activity and subcellular localization. The percentage of transformed mutant animals reverted to wild type is indicated and the number of animals scored is in brackets. Localization of the tagged mutant DPY-11 proteins within the expressing cells is shown on the right column. The GFP part is not to scale.