Figure 2.
PGL granules act as sites for mRNA metabolism that are distinct from stress granules. (A) Distinct poly(A) mRNA puncta are absent from somatic cells of WT embryos at 20°C, but are detected in germ precursor cells (highlighted in dashed circles) where they colocalize with P granules. PGL proteins are quickly degraded by autophagy in WT embryos under normal growth conditions. Thus, PGL granules are largely absent in embryos at late developmental stages. A few small granules are detected at early embryonic stages. One focal plane in a ∼100-cell-stage embryo is shown in A. (B) A large number of puncta containing poly(A) mRNAs accumulate in WT embryos at 26°C. These puncta are colocalized with P granules in germ precursor cells (Z2 and Z3, highlighted in dashed circles) and PGL granules in somatic cells labeled by PGL-1::TagRFP. (C) Poly(A) mRNA puncta are largely absent from PGL granules in somatic cells in atg-3 mutant embryos at 20°C but are detected in germ precursor cells (highlighted in dashed circles). (D) In atg-3 mutant embryos at 26°C, multiple poly(A) mRNA puncta accumulate, which are colocalized with P granules in germ precursor cells (highlighted in dashed circles) and PGL granules in somatic cells. One focal plane in a ∼100–200 cell-stage embryo is shown in B–D. The poly(A) mRNA puncta in atg-3 mutants at 20°C are much weaker in intensity. (E and F) Quantification of the percentage (%) of PGL granules positive for poly(A) mRNA (E), and the number of poly(A) mRNA puncta (F) in somatic cells per focal plane. Data are shown as mean ± SEM (n = 5; n refers to five images from the corresponding five embryos analyzed for each genotype or condition). Two-tailed, unpaired t test results: *P < 0.05, **P < 0.01, ***P < 0.001. (G) Quantification of the mean fluorescence intensity of somatic poly(A) mRNA puncta. Data are shown as mean ± SEM (n = 23, 14, and 37 puncta for WT embryos at 26°C, atg-3 mutant embryos at 20, and 26°C, respectively; n refers to the number of puncta analyzed for each genotype or condition). Two-tailed, unpaired t test results: ***P < 0.001. (H) GTBP-1::GFP forms no distinct granules, while PGL granules accumulate in WT embryos at the ∼100–200 cell stage at 26°C. (I and J) P bodies, detected by anti-DCAP-1, are separate from PGL granules in WT embryos at the ∼100–200 cell stage at 26°C. The germ precursor cells (Z2 and Z3) are highlighted in dashed circles. J shows quantification of the colocalization of DCAP-1 bodies with PGL granules. Data are shown as mean ± SEM (n = 3; n refers to three images from the corresponding three embryos analyzed). Scale bars: 5 µm for A–D, H, and I; 2 µm for enlarged images in A–D, H, and I.

PGL granules act as sites for mRNA metabolism that are distinct from stress granules. (A) Distinct poly(A) mRNA puncta are absent from somatic cells of WT embryos at 20°C, but are detected in germ precursor cells (highlighted in dashed circles) where they colocalize with P granules. PGL proteins are quickly degraded by autophagy in WT embryos under normal growth conditions. Thus, PGL granules are largely absent in embryos at late developmental stages. A few small granules are detected at early embryonic stages. One focal plane in a ∼100-cell-stage embryo is shown in A. (B) A large number of puncta containing poly(A) mRNAs accumulate in WT embryos at 26°C. These puncta are colocalized with P granules in germ precursor cells (Z2 and Z3, highlighted in dashed circles) and PGL granules in somatic cells labeled by PGL-1::TagRFP. (C) Poly(A) mRNA puncta are largely absent from PGL granules in somatic cells in atg-3 mutant embryos at 20°C but are detected in germ precursor cells (highlighted in dashed circles). (D) In atg-3 mutant embryos at 26°C, multiple poly(A) mRNA puncta accumulate, which are colocalized with P granules in germ precursor cells (highlighted in dashed circles) and PGL granules in somatic cells. One focal plane in a ∼100–200 cell-stage embryo is shown in B–D. The poly(A) mRNA puncta in atg-3 mutants at 20°C are much weaker in intensity. (E and F) Quantification of the percentage (%) of PGL granules positive for poly(A) mRNA (E), and the number of poly(A) mRNA puncta (F) in somatic cells per focal plane. Data are shown as mean ± SEM (n = 5; n refers to five images from the corresponding five embryos analyzed for each genotype or condition). Two-tailed, unpaired t test results: *P < 0.05, **P < 0.01, ***P < 0.001. (G) Quantification of the mean fluorescence intensity of somatic poly(A) mRNA puncta. Data are shown as mean ± SEM (n = 23, 14, and 37 puncta for WT embryos at 26°C, atg-3 mutant embryos at 20, and 26°C, respectively; n refers to the number of puncta analyzed for each genotype or condition). Two-tailed, unpaired t test results: ***P < 0.001. (H) GTBP-1::GFP forms no distinct granules, while PGL granules accumulate in WT embryos at the ∼100–200 cell stage at 26°C. (I and J) P bodies, detected by anti-DCAP-1, are separate from PGL granules in WT embryos at the ∼100–200 cell stage at 26°C. The germ precursor cells (Z2 and Z3) are highlighted in dashed circles. J shows quantification of the colocalization of DCAP-1 bodies with PGL granules. Data are shown as mean ± SEM (n = 3; n refers to three images from the corresponding three embryos analyzed). Scale bars: 5 µm for A–D, H, and I; 2 µm for enlarged images in A–D, H, and I.

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