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First published online 2 September 2008
doi: 10.1242/jcs.034678

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Rab11 is required for synchronous secretion of chondroitin proteoglycans after fertilization in Caenorhabditis elegans

¹ Laboratory of Molecular Traffic, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371-8512, Japan
² Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA

View larger version (78K): [in this window] [in a new window]   Fig. 1. CAV-1 bodies/cortical granules deliver chondroitin proteoglycans to extracellular space after fertilization. (A,B) The gonads of transgenic animals expressing CAV-1::GFP were stained with an anti-chondroitin antibody (A) or WGA-TRITC (B). CAV-1 bodies were stained with the anti-chondroitin antibody and WGA-TRITC, indicating that these structures contain chondroitin proteoglycans and mucin-like glycoproteins. (C) The gonads of N2 wild-type worms were stained with anti-chondroitin antibody and WGA-TRITC. The right panels are 4x enlargements of the boxed area indicated in merged images. Scale bars: 10 µm. (D) Stylized drawing of one gonad arm connected to the spermatheca and the uterus. Oocytes and embryos are numbered according to their positions from the spermatheca. Schematic localization of CAV-1::GFP is indicated in green. Chondroitin proteoglycans and mucin-like glycoproteins are indicated in orange. Developmental events are assembly-line-like processes and take place at indicated positions of the germline.

View larger version (81K): [in this window] [in a new window]   Fig. 2. RAB-11.1 and SYN-4 are required for exocytosis of cortical granules. The subcellular localization of CAV-1::GFP was determined in mock (A,B), rab-11.1(RNAi) (C,D), syn-4(RNAi) (E,F) and emb-27(RNAi) (G,H) animals by confocal laser microscopy. (A) In mock RNAi-treated animals, CAV-1::GFP gradually appears in ring-shaped structures (CAV-1 bodies/cortical granules, the inset) as oocytes grow. (B) The cortical granules are synchronously exocytosed in fertilized embryos and CAV-1::GFP targeted to the PM is then internalized for degradation within one-cell cycle. (C-F) RNAi of rab-11.1 or syn-4 causes accumulation of cortical granules around the nuclear envelope in oocytes (C,E, arrowheads) and inhibits fusion of cortical granules with the PM in embryos after fertilization (D,F). In the emb-27(RNAi) worm, the localization of CAV-1::GFP is normal in oocytes (G) but cortical granules remain in embryos (H). Oocytes and embryos are maturated from left to right. The numbers indicate the positions of oocytes and embryos from the spermatheca. The insets show 2x enlargements of the boxed area. Scale bars: 10 µm.

View larger version (119K): [in this window] [in a new window]   Fig. 3. Cortical granule exocytosis requires RAB-11.1 and SYN-4. (A,B) Cortical granule formation is not impaired by rab-11.1(RNAi) and syn-4(RNAi). The gonad of rab-11.1(RNAi) or syn-4(RNAi) animals were stained with WGA-TRITC. (C,D) Cortical granule exocytosis is inhibited in rab-11.1(RNAi) and syn-4(RNAi) embryos. Embryos were dissected out from the adult worms and stained with WGA-TRITC. Multinucleated embryos arrested at the one-cell stage are shown. The insets show 2x enlargements of the boxed area. Scale bars: 10 µm.

View larger version (84K): [in this window] [in a new window]   Fig. 4. Dynamic behavior of GFP::RAB-11.1 during oocyte-embryo transition. (A,B) Subcellular localization of GFP::RAB-11.1 in wild-type oocytes. GFP::RAB-11.1 localizes to the cortical region in growing oocytes (–3 and –2 oocytes) but transiently accumulates on the ring-like structures in the ovulating oocyte (–1 oocyte). Arrowheads indicate Golgi-like punctate structures deeper in the cytoplasm. Middle (A) and top (B) focal planes are shown. (C) Subcellular localization of GFP::RAB-11.1 in wild-type embryos. A projected image of confocal z-stacks is shown. GFP::RAB-11.1 localizes on the ring-shaped structures in the early one-cell stage embryo (+1 embryo). (D) Subcellular localization of GFP::RAB-11.1 in emb-27(RNAi) embryos. GFP::RAB-11.1 stably localizes on the ring-shaped structures in +1 and +2 embryos arrested at the one-cell stage. Some GFP::RAB-11.1 localizes on large cytoplasmic foci in +3 embryo (asterisks). Insets in A-D show 2x enlargements of the boxed areas. (E-H) GFP::RAB-11.1 localizes on cortical granules in wild-type one-cell stage embryos. Embryos were dissected from animals expressing GFP::RAB-11.1 and stained sequentially with WGA-TRITC and an anti-GFP antibody. Enlarged images of the boxed area in E are shown in F-H. Arrowheads show colocalization of GFP::RAB-11.1 and WGA. Scale bars: 10 µm in A-E; 2.5 µm in H. SP, spermatheca.

View larger version (130K): [in this window] [in a new window]   Fig. 5. Subcellular localization of SYN-4 and SNB-1 in oocytes and embryos. (A,B) The gonads of animals expressing CAV-1::GFP were stained with anti-SYN-4 (A) or anti-SNB-1 (B) antibodies. SYN-4 localizes mainly on the PM but a part of SYN-4 is detected on cortical granules (arrowheads in A). SNB-1 localizes on the cortical granules (arrowheads in B) and small cytoplasmic vesicles. (C) The gonads of wild-type animals were stained with anti-SNB-1 antibody and WGA-TRITC. SNB-1 localizes to cortical granules labeled with WGA (arrowheads). (D,E) Subcellular localization of GFP::SNB-1 in the germline. In oocytes, GFP::SNB-1 localizes to ring-shaped structures (D). Unlike CAV-1::GFP, GFP::SNB-1 is not degraded in embryos and the GFP signal is stably detected in developing embryos (E). The insets show 2x enlargements of the boxed areas. Scale bars: 10 µm.

View larger version (97K): [in this window] [in a new window]   Fig. 6. RAB-11.1 and EMB-27 are required for proper eggshell formation after fertilization. The permeability of embryos to FM4-64 was examined in mock (A), emb-27(RNAi) (B) and rab-11.1(RNAi) (C) animals expressing CAV-1::GFP. A newly fertilized wild-type embryo is permeable to FM4-64 (+1 embryo in A, an arrow indicates the PM stained with FM4-64) but blastomeres are not (+2 and +3 embryos in A). The emb-27(RNAi) and rab-11.1(RNAi) embryos are permeable to FM4-64. Nomarski image is on the right in each pair. Scale bar: 10 µm.

View larger version (89K): [in this window] [in a new window]   Fig. 7. Chitin layer formation is independent of cortical granule exocytosis. Embryos of animals expressing CAV-1::GFP were stained with rhodamine-conjugated chitin-binding domain (ChBD). (A) Mock; (B) rab-11.1(RNAi); (C) emb-27(RNAi). Nomarski images merged with fluorescent images are also shown on the right in each pair. Scale bars: 10 µm.

View larger version (130K): [in this window] [in a new window]   Fig. 8. Caveolins are not required for cortical granule biogenesis. The gonads were dissected and stained with WGA-TRITC. (A) Wild type; (B) cav-1(ok2089); and (C) cav-1(ok2089); cav-2(hc191). The insets show 1.5x enlargements of the boxed areas. Scale bar: 10 µm.

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