Analysis of P granules in vivo and ex vivo

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MAIN TEXT
Liquid-liquid phase separation (LLPS) is a spontaneous de-mixing process where molecules partition between condensed and dilute liquid phases 1,4 .LLPS has been proposed to underlie the formation of RNA granules in cells, and several RNA-binding proteins have been found to undergo LLPS in vitro 5 .Some LLPS condensates assembled in vitro mature over time into nondynamic, gel-like condensates [6][7][8][9] .Solidification of liquid phases in RNA granules has been linked to pathological states, as in certain neurodegenerative diseases where RNA granule components become trapped in non-dynamic aggregates 4,10 .Solid cores have also been observed in stress granules, but whether they are by-products of LLPS or essential structures that support granule assembly is not yet known 11,12 .Here, we have investigated a possible role for non-dynamic condensates in the formation of P granules, RNA granules that form under normal conditions in the germline of C. elegans.
P granules were the first cytoplasmic RNA granules reported to display liquid-like behaviors 2 .P granules are roughly round, can fuse with each other, and exchange components with the cytoplasm.P granules are perinuclear during most of germline development and become cytoplasmic at the oocyte-to-embryo transition 13 .During the first embryonic cell cycle, P granules disassemble and reassemble in the germ plasm, a region of cytoplasm in the posterior of the embryo that is partitioned to the germ lineage 2 (Fig. 1).Genetic analyses have identified two classes of proteins required for P granule assembly in embryos: the RGG-domain protein PGL-3 (and its paralog PGL-1), which form condensates that recruit other RNAbinding proteins 14,15 , and the intrinsicallydisordered protein MEG-3 (and its paralog MEG-4), which localizes and stabilizes PGL condensates in germ plasm 16,17 .PGL-3 and MEG-3 overlap only partially in P granules, with PGL-3 enriched in the center and MEG-3 enriched at the periphery 17 .Lattice light sheet microscopy revealed that MEG-3 forms a structured shell around a more amorphous PGL core, raising the possibility that the MEG and PGL phases have distinct material properties 17 .
Liquid phases can be sensitive to changes in temperature and concentration 20,21 .To test the effect of temperature, we filmed embryos expressing PGL-3::mCherry and MEG-3::meGFP while subjecting them to rapid temperature shifts using a temperature-controlled stage.An up-shift from 20 o C (normal growth temperature) to 34 o C caused PGL-3::mCherry to dissolve into the cytoplasm within seconds.Return to 20 o C caused  PGL-3::mCherry to reappear on the granules after ~ 8 min (Fig. 2a).MEG-3::meGFP, in contrast, remained in granules throughout the experiment.A longer 15 minute shift to 34 o C also did not affect MEG-3::meGFP distribution (Extended Data Fig. 2a).To test the effect of concentration, we punctured the eggshell of the embryo with a laser to release cellular contents into an aqueous buffer (Fig. 2b -See Methods for a detailed protocol that avoids technical artifacts that confounded initial experiments reported in the previous versions of this manuscript).Consistent with a liquid phase, PGL-3::mCherry diffused immediately upon dilution into the buffer.PGL-1::GFP also dissolved immediately ex vivo (Extended Data Fig. 2b).MEG-3::meGFP, in contrast, persisted in the granule phase, as the granules flowed out of the embryo into the dilute buffer (Fig. 2b and Supplemental Movie 3).On average, 64 ± 18% of MEG-3::meGFP fluorescence remained in the granule phase after extrusion (Fig. 2c).The extruded MEG-3::meGFP granules persisted in buffer for at least 1 hour (Extended Data Fig. 2c,d).MEG-3::meGFP granules remained stable when extruded into buffer containing 1M NaCl, 1,6 hexanediol (5%), or 10mM ATP, chemicals that dissolve liquid phases 22,23 (Fig. 2c).MEG-3::meGFP granules, however, dissolved in SDS (0.5%) (Fig. 2c).These observations suggest that the MEG-3 phase is not an irreversible aggregate, but a stable, gel-like phase.In contrast, the PGL phase behaves like a liquid.
P granules were the first RNA granules proposed to behave like liquid droplets suspended in the cytoplasm.Our observations indicate that the liquid phase of P granules is stabilized by a molecularly distinct enveloping layer that has gellike properties.Consistent with a gel-like phase, the MEG-3 shell is non-dynamic, resistant to dilution, high salt and aliphatic alcohol, and sensitive to SDS.MEG-3 is essential to assemble PGL droplets in the germ plasm 17 .The MEG-3 shell may therefore function as a protective "skin" that stabilizes the liquid core of P granules against factors in the cytoplasm that would otherwise dissolve the PGL phase.In embryos where P granules initiate cycles of disassembly and reassembly, the MEG-3 phase becomes dynamic in a manner that depends on DYRK/MBK-2 kinase.Assemblies that transition between gel and liquidlike dynamics have also been suggested to exist in nucleoli and stress granules, which solidify ex vivo and exhibit ATP-dependent dynamics in vivo 11,24 .Solidification of RNA granules has been linked to aging, ATP-deprived environments and certain neurodegenerative disorders 4,10 .Our findings suggest that gel phases are natural and essential stabilizing components of RNA granules that, like polymers in other self-organizing structures (e.g.cytoskeleton), can cycle between dynamic and nondynamic states 25,26 .Models that only consider equilibrium dynamics therefore, may not be sufficient to describe the properties of RNA granules in cells.

Strain maintenance and RNA-mediated interference:
C. elegans was cultured according to standard methods at 20°C unless otherwise stated 27 .Genome editing was performed using CRISPR/Cas9 as previously described 28 .Strains used in this study are listed in Table S1.mbk-2(RNAi) was performed by feeding 29 using plasmids from the Ahringer or Openbiosystems libraries 17 .HT115 bacteria transformed with feeding vectors were grown at 37°C in LB + ampicillin (100 µg/ml) for 5 hr, induced with 5 mM IPTG for 45 min, plated on NNGM (nematode nutritional growth media) + ampicillin (100 µg/ml) + IPTG (1 mM), and grown overnight at room temperature.Embryos were hatched on RNAi plates and allowed to feed for ~3-4 days For visualization of arrested oocytes, fog-2(q71) females, which do not produce sperm, were collected in the L4 stage and matured for 8 hours before imaging in 20 nM levamisole.For imaging of embryos, adult females were dissected in 1X ex vivo buffer (25 mM HEPES pH 7, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2) to release embryos which were mounted onto 2% agarose pads on glass slides.

Microscopy:
Images were captured with a Zeiss Axio Imager equipped with a Yokogawa spinning-disc confocal scanner and Slidebook v 6.0 software (Intelligent Imaging Innovations).Unless otherwise noted embryo and oocyte images are Z stack maximum projections using a Z step size of 1 μm, spanning the depth of the oocyte or embryos using a 60x objective.All image analyses were conducted using the Fiji imageprocessing package (http://fiji.sc/Fiji).

Fluorescence Recovery after Photobleaching (FRAP):
Photobleaching of MEG-3::meGFP and PGL-3::mCherry in oocytes or embryos was performed using a laser to bleach a region slightly larger than the area of the granule (2 μm diameter) in the center plane of the Z-stack.Images were acquired as Zstacks with 10 planes with a step size 1 µm (oocytes) or 11 planes with a step size of 0.6 µm (embryos) by imaging 50ms exposures in the 488 channel and 25ms (embryos) or 50ms (arrested oocytes) exposures in the 561 channel using a 63x objective.
Images were acquired every 3 or 5s during a recovery phase of 180-300s after photobleaching.Maximum projections of Z-stacks acquired as above were analyzed in Fiji.The total or mean fluorescence intensity of the area containing the granule was measured at each time point.Fluorescence recovery was corrected for background and normalized to the initial granule intensity using the equation: nI = (I-I bkg )/(I i -I bkgi ), where nI is the background corrected and normalized fluorescence intensity, I is the intensity of the FRAPed granule, I bkg is the fluorescence intensity of the unFRAPed cytoplasm, I i is the initial intensity of the FRAPed granule before bleaching, and I bkgi is the initial background intensity.For MEG-3::meGFP FRAP experiments in mbk-2(RNAi) embryos where amplitudes only just approached full recovery during the time frame of the experiment, final recovery amplitudes were limited to A rec = 1.

Temperature shifts:
Temperature shift experiments were performed on a fluidic temperature control stage which enabled rapid temperature shifts (CherryTemp™, CherryBiotech).Embryos were prepared as described for FRAP experiments.Images were acquired using a z step size of 1µm, spanning the entire depth of the embryo.Final images are Z-stack maximum projections.Images were taken every 15s broken into the following segments: 1 min at 20°C followed by a 3-15 min shift to 34°C and then returned to 20°C for 25 min.

Ex vivo Experiments:
All ex vivo buffers were prepared in 25 mM HEPES pH 7, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2 buffered to pH 7.0 with NaOH and HCl using a Mettler Toledo Seveneasy pH meter and a Fisherbrand TM Accumet TM glass body standard combination mercury free electrode.Embryos were dissected from adult hermaphrodites in ex vivo buffer and mounted on glass slides with 3% agarose pads made with 1X ex vivo buffer.Embryo contents were extruded by puncturing the eggshell near the anterior region of the germline blastomere using a 3i Ablate!TM laser system at 532 nm pulse setting with a power level of 155.Note: to generate reproducible results, we have found that it is essential to mount embryos on 3% agarose pads made with 1X ex vivo buffer, and to puncture the embryo at the edge of the anterior region of the germline blastomere.Aiming the laser too close to the P granules gave inconsistent results, possibly due to the heat generated from the laser.We also confirmed this by manually extruding P granules on a glass slide, without the use of the laser (data not shown).It is also important to verify the pH of the buffer after addition of any additives, such as nucleotides.All embryo images are Z stack maximum projections using a Z step size of 1 μm, spanning the entire depth of the embryo.Images were acquired using 50ms exposures in the 488 channel and/or 50ms exposures in the 561 channel every 10s using a 63x objective.For Extended Data Fig. 2b, images were taken every 15min for 60min following extrusion.
To quantify MEG-3::meGFP persistence in granules, photomicrographs acquired as described above were analyzed using ImageJ64.Background was subtracted from each image using a rolling ball radius of 50 pixels.Pixels were smoothed 1 time and a pixel brightness threshold was set to 50-255 and the total integrated density of the objects (MEG-3::meGFP granules) was quantified.Total fluorescence intensity was calculated before (IB) and after (IA) extrusion and used to calculate a fluorescence ratio (IA/IB).For some embryos, granules left the field of view and could not be counted.The IA /IB, therefore, is a minimal estimate of the fraction of MEG-3::meGFP that remains in the granule phase after extrusion.Graphing and data fitting: All data was plotted and statistical analysis was conducted using Graphpad Prism 7 software.Fitting of recovery curves in FRAP experiments was conducted using Kaleidagraph (Synergy) software.

Figure 1 .
Figure 1.P granules contain two phases that exhibit distinct dynamics.(A) Photomicrographs of arrested oocytes (top row) and a 1-cell embryo (bottom row) expressing MEG-3::meGFP and PGL-3::mCherry fusions (strain JH3602).In this and all other figures, embryos are oriented with its posterior end (germ plasm) to the right.(B) Graphs showing MEG-3::meGFP or PGL-3::mCherry fluorescence recovery after photobleaching (FRAP) in arrested oocytes or embryos.Granule intensity was measured every 5s for 300s before and after bleaching.Values were normalized to initial fluorescence intensity and plotted as an average from multiple oocytes or embryos.Error bars represent mean ± SEM. (C) Graphs showing recovery rates from FRAP experiments plotted in B or SF1B.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 stage and genotype tested.MEG-3 dynamics are slower than PGL-1 and PGL-3 dynamics under all conditions.Error bars represent mean ± SD.For data directly on the x-axis of each graph indicated by a "*", no recovery was measured.

Figure 2 .
Figure 2. Effect of temperature and dilution on the MEG-3 and PGL-3 phases.(A) Time-lapse photomicrographs of a two-cell embryo expressing MEG-3::meGFP and PGL-3::mCherry fusions (JH3602) and cultured at 20 o C, up-shifted to 34 o C for 1 minute, and downshifted back to 20 o C for 8 minutes.MEG-3 remains in granules throughout.PGL-3 disperses at 34°C and returns to P granules at 20 o C. Scale bar is 10 μm.(B) Time-lapse photomicrographs of a two-cell embryo expressing MEG-3::meGFP and PGL-3::mCherry fusions (JH3602) 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.MEG-3::GFP persists in the granule phase, whereas mCherry::PGL-3 becomes dispersed.Scale bar is 10 μm.(C) Graph showing the fraction of MEG-3::GFP retained in the granular phase after extrusion.Total GFP fluorescence from granules was measured before laser puncture (IB) and after laser puncture (IA).Means are indicated along with error bars representing ± SD.