Germline P Granules Are Liquid Droplets That Localize by Controlled Dissolution/Condensation

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Science  26 Jun 2009:
Vol. 324, Issue 5935, pp. 1729-1732
DOI: 10.1126/science.1172046

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  1. Fig. 1

    P granule localization is not due to cytoplasmic flow. (A) Fluorescent images of GFP::PGL-1 (green) superimposed on differential interference contrast (DIC) (red). Time relative to pronuclear meeting (pnm). A, anterior; P, posterior. (B and C) The movement of P granules is similar to the movement of yolk granules. (B) Cytoplasmic flow field from PIV analysis of a single embryo (blue DIC image) during symmetry breaking. Yellow arrows indicate flow direction and magnitude. (C) Maximum-intensity projection of confocal stacks of GFP::PGL-1 P granules in the one-cell embryo during symmetry breaking; first frame, –8 min, 7 s pnm; last frame, –3 min, 30 s pnm; P granules in center of embryo move posteriorly (red arrow), and P granules near cortex move anteriorly (green arrows). (D) Overlay of P granule trajectories (white) from five GFP::PGL-1 embryos. Trajectories crossing into the posterior are shown in red, and those crossing into the anterior are in green. (E) Probability distribution of the location perpendicular to the AP axis of P granules crossing the midpoint [yellow line in (D)] into anterior (green) versus posterior (red). (F) The average flux per embryo (mean Embedded Image SEM, n = 5) indicates negligible net flux.

  2. Fig. 2

    Spatiotemporal changes in P granule size. (A) P granules throughout the one-cell embryo are initially dissolving; blue and red traces are intensities of individual GFP::PGL-1–labeled P granules in the anterior and posterior, respectively. Trajectories in the middle (black) are omitted for clarity. (B) mex-5(RNAi) (n = 5 embryos, blue curve) abrogates the anterior dissolution seen in WT GFP::PGL-1 embryos (n = 8, red curve), whereas par-1(RNAi) (n = 6, green curve) gives rise to dissolution throughout the embryo, as with spd-5(RNAi) embryos before symmetry breaking (n = 8, black curve). Data are shown as the mean ± SEM. (C) Example anterior (A) and posterior (P) GFP::PGL-1–labeled P granule (each recentered), showing anterior dissolution, and posterior dissolution followed by condensation. (D) Time sequence of GFP::PGL-1 embryo treated with spd-5(RNAi) for >24 hours to delay symmetry breaking. P granules completely dissolve, but then re-form upon symmetry breaking. (E) Fluorescence intensity in anterior (A) versus posterior (P) regions of a confocal slice through the middle of the embryo, after complete P granule dissolution in spd-5(RNAi) GFP::PGL-1 embryos. Regions of measurements indicated (mean ± SEM, n = 8). (F) The growth rate of P granules in the embryo posterior (arrow).

  3. Fig. 3

    P granules behave like liquids. (A) Jetting P granule (red outline) from a dissected GFP::PGL-1 germline nucleus (lower left, not visible). Shear direction, white arrows. (B) Dripping P granules (red outline) from a dissected GFP::PGL-1 germ line. Nucleus (N), white line. (C) Time scale of drop breakup and fusion events in dissected germline and early embryos, as a function of droplet size. The black line is a linear fit, yielding a ratio of viscosity to surface tension (η/γ) ≈ 2 s/μm. (D) Fluorescence recovery after photobleaching (FRAP) of a large nuclear-associated GFP::PGL-1–labeled P granule from an eight-cell embryo (upper left sequence; top to bottom = 20 s). Kymograph is along the black line in left sequence. Red denotes high intensity and blue, background intensity. The intensity decreases in the unbleached region (fluorescence loss in photobleaching, FLIP) as the bleached region recovers. From exponential fits, in the bleached region τFRAP = 4.7 s, and in the unbleached region τFLIP = 5.7 s.

  4. Fig. 4

    Proposed mechanism of P granule localization. (A) Concentration of soluble components versus position along AP axis (posterior to right). Before symmetry breaking, the condensation point Csat (dashed black line) is high across the embryo. The cytoplasmic concentration of P granule components Ccyt (green line) is much lower than Csat, and the embryo is undersaturated with P granule components everywhere. (B) Undersaturation leads to dissolution of P granules (large green spheres) into diffusing components (small green circles). (C) Symmetry breaking decreases Csat in the posterior, below Ccyt. (D) Consequently, posterior P granules condense from soluble components, whereas anterior P granules continue dissolving. The spatial dependence of Csat arises from gradients in polarity proteins, including MEX-5 (gray) and PAR-1 (blue).

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