A gel phase promotes condensation of liquid P granules in Caenorhabditis elegans embryos

Abstract

RNA granules are subcellular compartments that are proposed to form by liquid-liquid phase separation (LLPS), a thermodynamic process that partitions molecules between dilute liquid phases and condensed liquid phases. The mechanisms that localize liquid phases in cells, however, are not fully understood. P granules are RNA granules that form in the posterior of Caenorhabditis elegans embryos. Theoretical studies have suggested that spontaneous LLPS of the RNA-binding protein PGL-3 with RNA drives the assembly of P granules. We find that the PGL-3 phase is intrinsically labile and requires a second phase for stabilization in embryos. The second phase is formed by gel-like assemblies of the disordered protein MEG-3 that associate with liquid PGL-3 droplets in the embryo posterior. Co-assembly of gel phases and liquid phases confers local stability and long-range dynamics, both of which contribute to localized assembly of P granules. Our findings suggest that condensation of RNA granules can be regulated spatially by gel-like polymers that stimulate LLPS locally in the cytoplasm.

Fig. 1 –. P granule proteins exhibit distinct dynamics

a. Photomicrographs of 4-cell embryos co-expressing tagged MEG-3::GFP and PGL-3::mCherry or expressing single GFP-tagged P granule proteins GLH-1::GFP, LAF-1::GFP, DEPS-1::GFP in wildtype embryos or embryos depleted of MEG-3/4 by RNAi (see Source Data for n values). Scale bar is 10 μm. b. Graph showing recovery rates from FRAP experiments plotted in Supplementary Fig. 1. Each symbol represents one FRAP experiment on one granule. The rate of fluorescence recovery (Methods) is plotted on the y-axis on a log scale. The x-axis indicates the P granule protein, genotype, and stage tested. fog-2 is a feminizing mutation used here to arrest oocytes in the absence of sperm. Error bars represent mean ± SEM calculated from independent experiments. n values are indicated in the x-axis label. For data points marked by “*”, no recovery was detected. Supplementary Fig. 1 shows recovery curves and photomicrographs of representative FRAP experiments. Source data are available in Supplementary Data Set 1.

Fig. 2 –. Effect of temperature on the MEG-3 and PGL-3 phases

Time-lapse photomicrographs of a 2-cell embryo co-expressing MEG-3::GFP and PGL-3::mCherry fusions and cultured at 20°C, up-shifted to 34°C for 1 minute, and downshifted back to 20°C for 8 minutes. Scale bar is 10 μm. Images are representative of 6 independent experiments.

Fig. 3 –. Effect of dilution on the MEG-3 and PGL-3 phases

a. Time-lapse photomicrographs of a 2-cell embryo co-expressing MEG-3::GFP and PGL-3::mCherry fusions before, immediately after (~1s), and 10s after laser puncture of the eggshell. Laser puncture causes the contents of the posterior blastomere to spill onto the slide mixing with the aqueous buffer. Scale bar is 10 μm. b. Graphs showing the fraction of MEG-3::GFP or PGL-3::mCherry retained in the granular phase after extrusion. Total GFP or mCherry fluorescence from granules was measured before laser puncture (I_B) and after laser puncture (I_A) and used to calculate a fluorescence ratio (I_A/I_B). Means are indicated along with error bars representing ± SD calculated from multiple embryos (see Source Data for n values). Source data for panel b are available in Supplementary Data Set 1.

Fig. 4 –. In vitro assembly of MEG-3 and PGL-3 condensates

a. Representative photomicrographs of MEG-3 and PGL-3 condensates after incubation in condensation buffer for the indicated times. MEG-3 was trace labeled with Dylight488 (green) and PGL-3 with Alexa647 (red) (Methods). Each image was adjusted separately to optimize visualization of small condensates in the first time point, and condensate substructure in the other time points. See Supplementary Fig. 4b for uniformly processed images. Images are representative of data quantified in b and c. Scale bars are 5 μm. b. Graphs showing the number condensates of MEG-3 or PGL-3 when incubated singly (gray) or together (green) at increasing time of incubation in condensate buffer. Each data set includes condensates from 16 images collected from 4 experimental replicates. Circles indicate the mean and bars represent the SD. c. Graphs showing the distribution of total intensity of individual condensates of MEG-3 or PGL-3 when incubated singly (gray boxes) or together (green boxes) at increasing time of incubation in condensate buffer. Each data set includes condensates from 16 images collected from 4 experimental replicates. Black lines indicate the mean, whiskers represent the entire range of the data, and bars represent the 90% confidence interval calculated from >500 condensates for each condition. d. Graph showing fluorescence recovery after partial photobleaching (FRAP) of condensates assembled as in (a) and incubated for 30 min in condensate buffer. Granule intensity of MEG-3 or PGL-3 individual condensates (left panels, n=8) or co-condensates (right panels, n=7) was measured every 3s for 300s before and after bleaching. Values were normalized to initial fluorescence intensity, corrected for photobleaching and plotted as an average. Error bars represent mean ± SD. Representative images are shown in the bottom panels. Scale bars are 1 μm. See Supplementary Fig. 4d for FRAP of fully bleached PGL-3 droplets. e. Representative photomicrographs of MEG-3 and PGL-3 co-condensates imaged in vivo in 2 or 4-cell embryos or assembled in vitro. Scale bars are 1 μm. Representative in vivo images were selected from 8 embryos. Representative in vitro condensates were selected from data quantified in b and c. Additional examples of MEG-3/PGL-3 condensates assembled in vivo are shown in Supplementary Fig. 5. Source Data for panels b, c and d are available in Supplementary Data Set 1.

Fig. 5 –. MEG-3 forms a stable scaffold around the PGL-3 liquid phase

a. Representative images of PGL-3 (red), MEG-3 (green) or MEG-3-IDR (green) co-assemblies incubated in condensation buffer or buffer with 1M NaCl. Scale bars are 2 μm. Images are representative of data quantified in b. b. Graphs showing the relative proportion of PGL-3 (right panels), MEG-3 (top left panels), or MEG-3-IDR (bottom left panels) in condensates [calculated as the ratio of fluorescence in condensates (I_C) over total fluorescence (I_T)]. The y-axis is on a log scale. Single condensates are in black and co-condensates are in green. Each data point represents the average from 4 images collected in one experiment. For data points indicated by “*”, no condensates were detected. Bars represent the mean ± SD. Source data for panel b are available in Supplementary Data Set 1.

Fig. 6 –. Model for regulation of P granule assembly by the MEG-3 gel condensates.

MEG-3 condensates associate dynamically with PGL-3 condensates. Polarization of the embryo enriches MEG-3 in the posterior cytoplasm. Preferential stabilization of PGL-3 condensates in the posterior creates an anterior-to-posterior flux of PGL-3 which destabilizes PGL-3 condensates in the anterior.