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First published online 30 January 2008
doi: 10.1242/dev.015552

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DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells

Caroline A. Spike^1,*, Jason Bader^1,, Valerie Reinke² and Susan Strome^1,,

¹ Department of Biology, Indiana University, Bloomington, IN 47405, USA.
² Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA.

View larger version (101K): [in this window] [in a new window]   Fig. 1. GFP::PGL-1 is mis-localized in deps-1 mutants. (A,B) GFP::PGL-1 localizes to P granules in a wild-type two-cell embryo (A) and in an adult hermaphrodite germ line (B). P granules (arrowheads) are cytoplasmic in the posterior cell of two-cell embryos (A), perinuclear in pachytene germ cells (lower half of B), and both cytoplasmic and perinuclear in oocytes (upper half of B). (C,D) GFP::PGL-1 localizes poorly to P granules in a deps-1(bn121) two-cell embryo (C) and hermaphrodite germ line (D). Single-section confocal images of the mid-regions of embryos and germ lines are shown. Equivalent exposure times and settings were used to image A and C, and B and D. Scale bars: 10 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 2. DEPS-1 associates with P granules. (A) The predicted Y65B4BL.2/deps-1 gene and locations of mutant alleles. Coding region is in gray and UTRs are in white. In blue is a 61 amino acid region containing 21 serines, 7 threonines, 8 arginines and 7 alanines. bn121 removes nucleotides 988-1172 (horizontal line) and results in a predicted transcript with a stop immediately after amino acid M246. bn113 mutates a conserved residue in the 3' splice site. Amino acid and nucleotide sequence positions are with respect to the predicted translation start site. (B) Western blot analysis using affinity-purified anti-DEPS-1 and anti--tubulin as a loading control. The arrowhead indicates the position of DEPS-1. The asterisk indicates a 120 kDa band present in wild type and in deps-1 mutants. Molecular mass of protein standards in kDa is on the right. (C-F) Anti-DEPS-1 staining pattern of wild type (C,D,F) and deps-1(bn121) (E) embryos at advancing stages of development (C, four cell; D,E 16 cell; F 100 cell). (G,H) DEPS-1::GFP is concentrated on P granules in 100-cell embryos (G) and in germ lines and oocytes (H). (I-N) DEPS-1 and PGL-3 co-localize on P granules in wild-type germ lines (I-K, surface of the pachytene region) and oocytes (L-N). Merged images show DEPS-1 in green and PGL-3 in red. Arrowheads and arrows indicate P granules and nuclei, respectively. H-K are single-section confocal images; all other images are confocal stacks. Scale bars: 10 µm. Images C-G and I-N use the scale bar in C.

View larger version (15K): [in this window] [in a new window]   Fig. 3. deps-1 mutants display temperature-sensitive sterility and embryonic lethality. (A) Fertility profiles of deps-1(bn121) M-Z- at 15, 20 and 24.5°C. Mutant hermaphrodites were picked to individual plates as L4s, and the fertility of each animal scored several days later. Animals that generated (a) >20 or (b) 1-20 larval progeny are defined as fertile. Animals that did not have larval progeny but (c) laid dead eggs, (d) produced oocytes or oocyte-like material, or (e) failed to make either embryos or oocytes are defined as sterile. Numbers of adults examined were 45 (15°C), 132 (20°C) and 105 (24.5°C). (B) Number and quality of eggs laid by deps-1(bn121) M+Z- and M-Z- adults at 15 and 20°C and deps-1(bn121) M+Z- adults at 24.5°C. n.d., not determined.

View larger version (25K): [in this window] [in a new window]   Fig. 4. DEPS-1 promotes the accumulation of glh-1 and rde-4 mRNA and protein. (A-D) Wild-type and deps-1(bn124) germ lines stained with PA3, a marker for chromatin (A,C), and anti-GLH-1 (B,D). A-D are stacks of three confocal sections taken at 1 µm intervals showing germ nuclei present on the surface of the pachytene region of the hermaphrodite germ line. Scale bar: 10 µm. (E) Western blot analysis (top panel) of GLH-1 in whole worm extracts, with -tubulin as a loading control, and histogram (lower panel) of glh-1 mRNA (measured by quantitative RT-PCR) and GLH-1 protein levels in deps-1 mutants compared with wild type. (F) Similar analysis of rde-4 mRNA and RDE-4 protein levels, but using partially purified worm extracts, and comparing with rde-4 mutants and with wild type. The histograms show average ratios obtained in two independent experiments. The wild-type ratio was set at 1.0 in each experiment. For mutants, error bars indicate the s.e.m.

View larger version (10K): [in this window] [in a new window]   Fig. 5. deps-1 mutants are resistant to RNAi of the germline-expressed gene pos-1. (A) pos-1(RNAi) causes highly penetrant embryonic lethality among the progeny of wild-type animals but not deps-1 M+Z- or pgl-1 M-Z- mutants raised at 20°C. The number of dead embryos/total embryos is indicated on the right. (B) unc-52(RNAi) causes the progeny of wild-type and deps-1, but not rde-4, mutants to become paralyzed as adults. The number of paralyzed/total worms is on the right.

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