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. 2014 Dec 23;3:e04591. doi: 10.7554/eLife.04591

Figure 5. MEG-3 localizes to a dynamic domain that surrounds and penetrates the P granules.

(A) Gonad of an adult hermaphrodite expressing the GFP::MEG-3 transgene under the control of the meg-3 promoter and 3′ UTR. GFP::MEG-3 associates strongly with P granules only in embryos. (B) Fixed wild-type zygote stained with anti:MEG-3 and anti-PGL-1 sera. Inset: magnification of merged image. Arrow points to a MEG-3-positive/PGL-1-negative granule. (C) Live wild-type and pgl-1(RNAi);pgl-3(bn104) zygotes expressing GFP::MEG-3. GFP::MEG-3 localizes to a cytoplasmic gradient and P granules. P granule localization is lost in pgl-1(RNAi);pgl-3(bn104) zygotes, but the cytoplasmic gradient remains. (D) Still images from a movie acquired using lattice light sheet microscopy (Video 5). Max intensity projection of a Z stack through a pair of fusing granules. Time in seconds (Video 5) is indicated above each panel. GFP::MEG-3 and mCherry:PGL-3 domains are not completely co-localized. Also see Figure 5—figure supplement 3B. Resolution is 238 nm × 238 nm × 500 nm. Scale bars: upper left = 500 nm. (E) Lattice light sheet 3D-SIM mode reconstruction of GFP::MEG-3 in P granules in a living zygote (also see Video 6). Scale bars: upper left = 500 nm. First granule on the left: acquisition time is 1 s (Video 6). Subsequent granules: acquisition time is 1.7 s (Video 7).

DOI: http://dx.doi.org/10.7554/eLife.04591.018

Figure 5.

Figure 5—figure supplement 1. GFP::MEG-3 rescues meg-3 mutant and the MEG-3 antibody is specific.

Figure 5—figure supplement 1.

(A) Fixed zygotes stained for P granules (K76 antibody, red). Note the oocyte granules (arrows) in meg-3(tm4259) which are absent in the rescued strain meg-3(tm4259);GFP::MEG-3. At least 20 mothers were stained for each genotype and these images are representative. We could not test whether GFP::MEG-3 also rescues the P granule assembly defect of meg-3(tm4259) mutants, since this defect can only be scored using GFP::PGL-1 (antibody staining of P granules is not reliable enough for accurate counts at this stage). (B) Fixed meg-3(tm4259) zygote stained for P granules (K76 antibody, red) and with anti-MEG-3 serum. Anti-MEG-3 serum does not stain granules in meg-3(tm4259) embryos, but does so in wild-type (Figure 5B), demonstrating specificity of the serum.
DOI: http://dx.doi.org/10.7554/eLife.04591.019
Figure 5—figure supplement 2. MEG-4 localizes to P granules and MEG-3 and MEG-4 assemblies persist longer in disassembling granules than PGL-1.

Figure 5—figure supplement 2.

(A) Embryos expressing MEG-4::FLAG (endogenous locus) and stained with anti-FLAG antibody. (B) Top row: maximum projection image of embryo expressing MEG-4::FLAG (endogenous locus) and stained with anti-FLAG antibody and anti-PGL-1 (K76) antibody. Note the MEG-4:FLAG-positive/PGL-1 negative granules in the anterior part of the cell (where P granules disassemble). Bottom row: maximum projection of image of wild-type embryo (no MEG-4::FLAG) stained with anti-FLAG antibody and anti-PGL-1 (K76) antibody. Note the lack of FLAG staining confirming the specificity of the FLAG antibody. (C) Time-lapse of a disassembling P granule from an embryo expressing GFP::MEG-3 and mCherry::PGL-3 granule. During granule disassembly, GFP::MEG-3 persists for longer than mCherry::PGL-3. Also see Video 4. (D) Zygotes expressing GFP::MEG-3 imaged at mitosis. Genotypes are: pptr-1(RNAi) pptr-2(RNAi) and mbk-2(RNAi). Note the perduring GFP::MEG-3 clusters in the pptr-1pptr-2 zygotes which disassemble all PGL clusters by this stage (Figure 4A).
DOI: http://dx.doi.org/10.7554/eLife.04591.020
Figure 5—figure supplement 3. MEG-3 and PGL-1/3 overlap only partially in P granules.

Figure 5—figure supplement 3.

(A) Confocal images of two P granules in a fixed zygote expressing GFP::MEG-3. Endogenous PGL-1 is visualized using the K76 antibody. In all fixed preparations, GFP::MEG-3 never co-localized perfectly with PGL-1, but the appearance of the PGL-1 signal varied between experimental replicates. (B) Single plane images of GFP::MEG-3 and mCherry::PGL-3 in fused granule is also shown in Figure 5D. Time points are indicated above in seconds. Four consecutive z planes are shown with 0.3 μm z steps. (C) Graph plotting area of mCherry::PGL-3 signal (x axis) vs area of GFP::MEG-3 signal (y axis) for 37 granules from two embryos visualized by lattice light sheet microscopy (dithered mode). Each dot represents an individual granule. Diagonal lines indicate 1× and 1.5× fold increase of GFP::MEG-3 area over mCherry::PGL-3 area. In 34 of 37 granules, the GFP::MEG-3 signal occupied a larger area than the mCherry::PGL-3 signal (1.57× increase on average). The area occupied by the mCherry::PGL-3 signal was always entirely contained within the area occupied by the GFP::MEG-3 signal (37/37 granules analyzed).
DOI: http://dx.doi.org/10.7554/eLife.04591.021
Figure 5—figure supplement 4. MEG-1 only partially co-localizes with PGL-1.

Figure 5—figure supplement 4.

Fixed embryos imaged using confocal spinning disk microscopy. Top: endogenous MEG-1 antibody (green), K76 (anti-PGL-1, red). Middle: K76 (anti-PGL-1, green), GLH-2 (red). Bottom: GFP::MEG-3 (green), endogenous MEG-1 antibody (red).
DOI: http://dx.doi.org/10.7554/eLife.04591.022
Figure 5—figure supplement 5. Lattice light sheet microscopy identifies sub-granular MEG-3 domain.

Figure 5—figure supplement 5.

(A and B) Single plane images of granules from live embryos expressing GFP::MEG-3 imaged by lattice light sheet 3D-SIM. Granule in B is the same as in Video 6 and Figure 5E. Top row: each structured illumination phase is shown. Bottom row, left: maximum intensity image of the raw phase data. Bottom row, right: image after reconstruction. Note that the structure seen in the reconstructed images is present in the raw maximum images. (C) Top: maximum projections of lattice light sheet images of GFP::MEG-3 and mCherry::PGL-3 granules. MEG-3 sub-granular structure is preserved over successive frames. Bottom: maximum projections of lattice light sheet images of cytoplasmic GFP::MEG-3 and mCherry::PGL-3. Image contrast was enhanced to the same extent for both channels. mCherry::PGL-3 is not visible in the cytoplasm under these conditions. In contrast, GFP::MEG-3 is easily detected and is distributed non-uniformly. The partial pattern repetition seen in the three successive time points and the high signal-to-noise ratio (D) suggest that the cytoplasmic MEG-3 pattern represents true structure and not noise. (D) Photon counts and signal to noise for each movie.
DOI: http://dx.doi.org/10.7554/eLife.04591.023