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First published online September 29, 2004
doi: 10.1242/10.1242/jcs.01387

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Cathepsin L protease (CPL-1) is essential for yolk processing during embryogenesis in Caenorhabditis elegans

Department of Veterinary Parasitology, Institute of Comparative Medicine, University of Glasgow, Garscube Estate, Bearsden Road, Glasgow, G61 1QH, UK

View larger version (13K): [in a new window]   Fig. 1. Caenorhabditis elegans cathepsin L protease mutant cpl-1(ok360) phenotype could be rescued efficiently by extrachromosomal transformation with the wild-type Ce-cpl-1 gene. Western blot of wild-type N2 (lane 1), rescued homozygous cpl-1 mutant (lane 2) and non-rescued homozygous cpl-1 mutant (lane 3) adult worm extracts probed with anti-CPL-1 pro-region antibody. The upper band shows the wild-type protein (approximately 36 kDa) and lower band shows the mutant CPL-1 protein (approximately 22 kDa).

View larger version (81K): [in a new window]   Fig. 2. Caenorhabditis elegans cpl-1(ok360) embryos fail to undergo morphogenesis and accumulate large cytoplasmic vesicles. (A-D) Development of the wild-type embryo: 2-cell (A); 12-cell (B); 100-cell (C); and threefold (D) stage embryos. (E-I) Development of cpl-1(ok360) embryo: 2-cell (E), 8-cell (F); 28-cell (G) 100-cell (H); and terminal (I) stage embryos. Images B and F; C and G; and D and H; were taken at 30 minutes, 120 minutes and 7 hours, respectively, after division to the 2-cell stage. (I) Terminal mutant phenotype image was taken 15 hours after that in H. Arrowheads indicate abnormally large cytoplasmic vesicles in cpl-1(ok360) embryos. Each embryo is approximately 50 µm in length.

View larger version (76K): [in a new window]   Fig. 3. LysoSensor Green accumulates in large vesicles in Caenorhabditis elegans cpl-1(ok360) mutant embryos. (A-D) Wild-type embryos with LysoSensor Green in punctate cytoplasmic vesicles. (E-H) cpl-1(ok360) mutant embryos with LysoSensor Green in large cytoplasmic vesicles: 1-cell (A,E); 2-cell (B,F); 16 cell (C,G); 100-cell (D,H) stage embryos.

View larger version (95K): [in a new window]   Fig. 4. Loss of fluorescence from YP::GFP reporter gene and accumulation of YP170 in Caenorhabditis elegans cpl-1 mutant embryos. YP170::GFP fluorescence in oocytes and embryos in the gonad of wild-type (A) and cpl-1 mutant (C) hermaphrodites. B and D are Nomarski images of A and C, respectively. The vulva is indicated by an arrow. YP170::GFP in wild-type embryo at approximately 28-cell stage (E) and threefold stage (F). Significantly decreased levels of fluorescence are observed in similarly aged cpl-1(ok360) embryos (H,I). Immunofluorescence with antibody to YP170 in wild-type (G) and cpl-1 mutant (J) embryos. Each embryo is approximately 50 µm in length.

View larger version (140K): [in a new window]   Fig. 5. Immuno electron microscopy (IEM) on wild-type and cpl-1(ok360) adult Caenorhabditis elegans hermaphrodites with YP170 antibody. (A) Antibody localises to small yolk platelets in wild-type embryos. (B) Higher magnification of wild-type embryo. (C) Mutant (ok360) embryo showing accumulation of enlarged yolk platelets. Smaller yolk vesicles can also be observed in some embryonic cells. (D) Higher magnification of enlarged yolk vesicles in cpl-1(ok360) embryo. (E,F) Double labelling of yolk platelets with anti-pro-CPL-1 antibody (detected with 10 nm gold; arrowheads) and anti-YP170 antibody (detected with 15 nm gold; arrows) in a wild-type embryo (E) and in the hermaphrodite pseudocoelom (F). Bar, 500 nm.

View larger version (73K): [in a new window]   Fig. 6. Cytoplasmic vesicles induced by rab7 RNAi form before Caenorhabditis elegans cpl-1(ok360) mutant vesicles. (A,C) YP170::GFP accumulates in slightly enlarged cytoplasmic vesicles in rab7(RNAi) embryos (A) and rab7(RNAi)/cpl-1(ok360) double mutant embryos (C). The fluorescent vesicles are of similar size, but less abundant in the double mutant (C). (B,D) Immunofluorescence with YP170 antibody in rab7(RNAi) embryo (B) and cpl-1(ok360)/rab7(RNAi) double mutant embryo (D). Arrowheads indicate the abnormally large yolk vesicles that accumulate only in the double mutant.

View larger version (84K): [in a new window]   Fig. 7. YP170 and yolk receptor RME-2 accumulation in Caenorhabditis elegans cpl-1(ok360) mutant embryos. (A-F) Immunofluorescence with antibody to YP170 (red) and RME-2 (green) in wild-type (A-C) and cpl-1 mutant (D-F) embryos. Localisation of RME-2 to the periphery of enlarged yolk vesicles is seen in cpl-1 mutant embryos (F), but not in wild-type embryos (C). (B,C) The embryo on the right shows anti-RME-2 localisation in a one-cell stage wild-type embryo, prior to RME-2 degradation. (G,H) IEM localisation of RME-2 antibody to small vesicles in wild-type embryo (G) and to electron-light regions at the periphery of enlarged yolk vesicles in cpl-1(ok360) embryo (H). Bar, 500 nm.

View larger version (72K): [in a new window]   Fig. 8. RNAi of yolk receptor RME-2 partially rescues Caenorhabditis elegans cpl-1(ok360) mutant phenotype. (A) Development and morphogenesis in cpl-1(ok360) embryos following feeding on rme-2 RNAi plates. The positioning of birefringent gut granules (arrowheads) indicates normal gut development in the double mutant. (B) No morphogenesis is observed in single cpl-1(ok360) mutant embryos.