Cytoplasmic Partitioning of P Granule Components Is Not Required to Specify the Germline in
            <i>C. elegans</i>

Gallo, Christopher M.; Wang, Jennifer T.; Motegi, Fumio; Seydoux, Géraldine

doi:10.1126/science.1193697

Cited by 124 publications

(180 citation statements)

References 21 publications

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“…P granules are implicated in germ-line establishment and maintenance, but their precise function remains largely unknown. A recent study has shown that segregation of P granules to progenitor germ cells is only necessary for germ-line specification under certain conditions, suggesting a potential role for P granules in protection from stress (34); consistent with this, recent work has implicated LAF-1 and its close homolog VBH-1 in the stress response of C. elegans (35). P granules likely function in cytoplasmic RNA regulation, including storage and release of mRNA transcripts (36,37).…”

Section: Discussionmentioning

confidence: 57%

The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

Elbaum‐Garfinkle

Kim

Szczepaniak

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

1,152

1,357

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P granules and other RNA/protein bodies are membrane-less organelles that may assemble by intracellular phase separation, similar to the condensation of water vapor into droplets. However, the molecular driving forces and the nature of the condensed phases remain poorly understood. Here, we show that the Caenorhabditis elegans protein LAF-1, a DDX3 RNA helicase found in P granules, phase separates into P granule-like droplets in vitro. We adapt a microrheology technique to precisely measure the viscoelasticity of micrometer-sized LAF-1 droplets, revealing purely viscous properties highly tunable by salt and RNA concentration. RNA decreases viscosity and increases molecular dynamics within the droplet. Single molecule FRET assays suggest that this RNA fluidization results from highly dynamic RNA-protein interactions that emerge close to the droplet phase boundary. We demonstrate than an N-terminal, arginine/glycine rich, intrinsically disordered protein (IDP) domain of LAF-1 is necessary and sufficient for both phase separation and RNA-protein interactions. In vivo, RNAi knockdown of LAF-1 results in the dissolution of P granules in the early embryo, with an apparent submicromolar phase boundary comparable to that measured in vitro. Together, these findings demonstrate that LAF-1 is important for promoting P granule assembly and provide insight into the mechanism by which IDP-driven molecular interactions give rise to liquid phase organelles with tunable properties.liquid droplets | intracellular phase transition | intrinsically disordered proteins | RNA granules I ntracellular RNA/protein (RNP) assemblies, including germ granules, processing bodies, stress granules, and nucleoli, are key players in the regulation of gene expression (1). RNP bodies, also referred to as RNA granules, function in diverse modes of RNA processing, including splicing, degradation, and translational repression of mRNA. These ubiquitous structures lack a membrane boundary but nonetheless represent a coherent organelle composed of thousands of molecules, manifesting as microscopically visible puncta in both the cytoplasm and the nucleus.Recent studies have demonstrated the apparent liquid-like behavior of various RNP bodies (2-5) including wetting, dripping, and relaxation to spherical structures upon fusion or shearing. The assembly and disassembly of liquid-like organelles appears to be governed by a phase separation process, demonstrated by a concentration-dependent condensation/dissolution of P granules (2, 6) and the assembly and size scaling of the nucleolus (7) in the Caenorhabditis elegans embryo. Liquid phase separation has also been suggested to play a role in stress granule assembly (4) and in multivalent signaling proteins (8). These studies lend increasing support to the hypothesis that liquid phases play a central role in intracellular organization. However, the specific molecular interactions that drive phase separation and the mechanisms by which liquid properties impart cellular function remain largely unclear. P granule...

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Section: Discussionmentioning

confidence: 57%

The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

Elbaum‐Garfinkle

Kim

Szczepaniak

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

1,152

1,357

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“…These were labeled with a GFP-PGL-1 fluorescent fusion protein (Gallo et al, 2010). Images were acquired in 3D at 10-second intervals from metaphase until the one-cell embryo had divided.…”

Section: Chromatin Rotates Around the Anterior-posterior Axis During mentioning

confidence: 99%

Live imaging reveals spatial separation of parental chromatin until the four-cell stage in Caenorhabditis elegans embryos

Bolková

Lanctôt²

2016

Int. J. Dev. Biol.

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The parental genomes are initially spatially separated in each pronucleus after fertilization. Here we have used green-to-red photoconversion of Dendra2-H2B-labeled pronuclei to distinguish maternal and paternal chromatin domains and to track their spatial distribution in living Caenorhabditis elegans embryos starting shortly after fertilization. Intermingling of the parental chromatin did not occur until after the division of the AB and P1 blastomeres, at the 4-cell stage. Unexpectedly, we observed that the intermingling of chromatin did not take place during mitosis or during chromatin decondensation, but rather ~3-5 minutes into the cell cycle. Furthermore, unlike what has been observed in mammalian cells, the relative spatial positioning of chromatin domains remained largely unchanged during prometaphase in the early C. elegans embryo. Live imaging of photoconverted chromatin also allowed us to detect a reproducible 180° rotation of the nuclei during cytokinesis of the one-cell embryo. Imaging of fluorescently-labeled P granules and polar bodies showed that the entire embryo rotates during the first cell division. To our knowledge, we report here the first live observation of the initial separation and subsequent mixing of parental chromatin domains during embryogenesis.

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“…For example, P-granules are transported along the cytoplasm (8), but it was demonstrated that the flow does not contribute to asymmetric localization of P-granules (11). Cytoplasmic flow might be important for the asymmetric localization of other components that localize posteriorly at this stage, or may stimulate association of cytoplasmic material with the posterior cortex (8,12).…”

mentioning

confidence: 99%

Hydrodynamic property of the cytoplasm is sufficient to mediate cytoplasmic streaming in the Caenorhabiditis elegans embryo

Niwayama

Shinohara

Kimura

2011

Proc. Natl. Acad. Sci. U.S.A.

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Cytoplasmic streaming is a type of intracellular transport widely seen in nature. Cytoplasmic streaming in Caenorhabditis elegans at the one-cell stage is bidirectional; the flow near the cortex ("cortical flow") is oriented toward the anterior, whereas the flow in the central region ("cytoplasmic flow") is oriented toward the posterior. Both cortical flow and cytoplasmic flow depend on non-musclemyosin II (NMY-2), which primarily localizes in the cortex. The manner in which NMY-2 proteins drive cytoplasmic flow in the opposite direction from remote locations has not been fully understood. In this study, we demonstrated that the hydrodynamic properties of the cytoplasm are sufficient to mediate the forces generated by the cortical myosin to drive bidirectional streaming throughout the cytoplasm. We quantified the flow velocities of cytoplasmic streaming using particle image velocimetry (PIV) and conducted a threedimensional hydrodynamic simulation using the moving particle semiimplicit method. Our simulation quantitatively reconstructed the quantified flow velocity distribution resolved through PIV analysis. Furthermore, our PIV analyses detected microtubule-dependent flows during the pronuclear migration stage. These flows were reproduced via hydrodynamic interactions between moving pronuclei and the cytoplasm. The agreement of flow dynamics in vivo and in simulation indicates that the hydrodynamic properties of the cytoplasm are sufficient to mediate cytoplasmic streaming in C. elegans embryos. fluid dynamics | particle method | Newtonian fluid | cell polarity | algae

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Cytoplasmic Partitioning of P Granule Components Is Not Required to Specify the Germline in C. elegans

Cited by 124 publications

References 21 publications

The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

Live imaging reveals spatial separation of parental chromatin until the four-cell stage in Caenorhabditis elegans embryos

Hydrodynamic property of the cytoplasm is sufficient to mediate cytoplasmic streaming in the Caenorhabiditis elegans embryo

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