Dual Specificity Kinase DYRK3 Couples Stress Granule Condensation/Dissolution to mTORC1 Signaling

Wippich, Frank; Bodenmiller, Bernd; Trajkovska, Maria; Wanka, Stefanie; Aebersold, Ruedi; Pelkmans, Lucas

doi:10.1016/j.cell.2013.01.033

Cited by 538 publications

(571 citation statements)

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“…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.…”

supporting

confidence: 59%

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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.

1,150

1,292

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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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supporting

confidence: 59%

“…An increasing body of work suggests that P granules and other RNP bodies assemble by a type of intracellular phase transition (2,4,7,19). However, molecular-level insight into the components necessary to drive phase separation and maintain liquidlike properties is severely lacking.…”

Section: Discussionmentioning

confidence: 99%

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.

1,150

1,292

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“…The mechanisms by which such structures form and stably persist are not well understood. However, recent evidence suggests that these membraneless organelles, such as P granules (1,2), nucleoli (3), and stress granules (4), are liquid-phase droplets that may assemble by intracellular phase separation (5,6). This concept is supported by work on synthetic systems, including repetitive protein domains that form droplets in vitro and in the cytoplasm (7) and intrinsically disordered protein domains that show signatures of liquid-liquid phase separation when expressed in the cell nucleus (8).…”

mentioning

confidence: 68%

RNA transcription modulates phase transition-driven nuclear body assembly

Berry

Weber

Vaidya

et al. 2015

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

468

426

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Nuclear bodies are RNA and protein-rich, membraneless organelles that play important roles in gene regulation. The largest and most well-known nuclear body is the nucleolus, an organelle whose primary function in ribosome biogenesis makes it key for cell growth and size homeostasis. The nucleolus and other nuclear bodies behave like liquid-phase droplets and appear to condense from the nucleoplasm by concentration-dependent phase separation. However, nucleoli actively consume chemical energy, and it is unclear how such nonequilibrium activity might impact classical liquid-liquid phase separation. Here, we combine in vivo and in vitro experiments with theory and simulation to characterize the assembly and disassembly dynamics of nucleoli in early Caenorhabditis elegans embryos. In addition to classical nucleoli that assemble at the transcriptionally active nucleolar organizing regions, we observe dozens of "extranucleolar droplets" (ENDs) that condense in the nucleoplasm in a transcription-independent manner. We show that growth of nucleoli and ENDs is consistent with a first-order phase transition in which late-stage coarsening dynamics are mediated by Brownian coalescence and, to a lesser degree, Ostwald ripening. By manipulating C. elegans cell size, we change nucleolar component concentration and confirm several key model predictions. Our results show that rRNA transcription and other nonequilibrium biological activity can modulate the effective thermodynamic parameters governing nucleolar and END assembly, but do not appear to fundamentally alter the passive phase separation mechanism.RNA/protein droplets | intracellular phase separation | Brownian coalescence | Ostwald ripening | Flory-Huggins regular solution theory L iving cells are composed of complex and spatially heterogeneous materials that partition into functional compartments called organelles. Many organelles are vesicle-like structures with an enclosing membrane. However, a large number of RNA and protein-rich bodies maintain a dynamic but coherent structure even in the absence of a membrane. These so-called RNA/ protein granules are found in the cytoplasm and in the nucleus, where they are referred to as nuclear bodies. The mechanisms by which such structures form and stably persist are not well understood. However, recent evidence suggests that these membraneless organelles, such as P granules (1, 2), nucleoli (3), and stress granules (4), are liquid-phase droplets that may assemble by intracellular phase separation (5, 6). This concept is supported by work on synthetic systems, including repetitive protein domains that form droplets in vitro and in the cytoplasm (7) and intrinsically disordered protein domains that show signatures of liquid-liquid phase separation when expressed in the cell nucleus (8).By examining the cell size-dependent assembly of nucleoli in early Caenorhabditis elegans embryos, we previously showed that nucleolar assembly is controlled by a concentration-dependent phase transition (9). Using a simple model based on the de...

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“…More recent studies have suggested that the activities of P-bodies, and other RNP granules, may be determined by the presence of the more peripheral proteins associated with these structures. For example, these cytoplasmic granules have been found to influence cell physiology by recruiting signaling molecules important for the normal regulation of growth and survival (Arimoto et al 2008;Takahara and Maeda 2012;Kedersha et al 2013;Thedieck et al 2013;Wippich et al 2013;. The reasons for this recruitment are likely manifold and similar to those that apply to the membrane-bound compartments in the cytoplasm.…”

mentioning

confidence: 99%

The Activity-Dependent Regulation of Protein Kinase Stability by the Localization to P-Bodies

Zhang¹,

Shi²,

Varia³

et al. 2016

Genetics

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The eukaryotic cytoplasm contains a variety of ribonucleoprotein (RNP) granules in addition to the better-understood membrane-bound organelles. These granules form in response to specific stress conditions and contain a number of signaling molecules important for the control of cell growth and survival. However, relatively little is known about the mechanisms responsible for, and the ultimate consequences of, this protein localization. Here, we show that the Hrr25/CK1d protein kinase is recruited to cytoplasmic processing bodies (P-bodies) in an evolutionarily conserved manner. This recruitment requires Hrr25 kinase activity and the Dcp2 decapping enzyme, a core constituent of these RNP granules. Interestingly, the data indicate that this localization sequesters active Hrr25 away from the remainder of the cytoplasm and thereby shields this enzyme from the degradation machinery during these periods of stress. Altogether, this work illustrates how the presence within an RNP granule can alter the ultimate fate of the localized protein.KEYWORDS ribonucleoprotein granules; processing bodies; protein kinase; protein stability; Dcp2 decapping enzyme; casein kinase 1 T HE eukaryotic cell is subdivided into distinct functional areas by the presence of a variety of organelles. The best understood of these are the membrane-bound structures, like the nucleus, endoplasmic reticulum, and mitochondria. These traditional compartments are relatively stable and essential for the proper compartmentalization of the different reactions occurring in the cytoplasm. However, the cell also contains a collection of nonmembraneous organelles that are more dynamic in nature and form in response to particular cellular and environmental stimuli. Perhaps the two best characterized of these are the centrosome and nucleolus, which act as a microtubule-organizing center and a subnuclear site of ribosome assembly, respectively (Greenan et al. 2010;Brangwynne 2011;Brangwynne et al. 2011). This latter class also includes a number of recently identified cytoplasmic ribonucleoprotein (RNP) granules like the processing body (P-body) and stress granule (Anderson and Kedersha 2009;Balagopal and Parker 2009;Thomas et al. 2011). These cytoplasmic structures have been conserved through evolution and have been linked to a variety of human diseases, including certain neurodegenerative disorders, cancers, and autoimmune conditions (Li et al. 2013;Anderson et al. 2015). Despite this importance to human health, relatively little is known about the manner in which these RNP granules influence biological processes in the cell.These cytoplasmic RNP granules typically assemble in response to environmental stress or particular developmental cues (Thomas et al. 2011). Granule formation occurs as a consequence of the regulated coalescence of specific sets of proteins and translationally repressed mRNAs at discrete sites in the cytoplasm (Anderson and Kedersha 2009;Balagopal and Parker 2009). The presence of these core proteins often depends upon specific RNA-...

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Dual Specificity Kinase DYRK3 Couples Stress Granule Condensation/Dissolution to mTORC1 Signaling

Cited by 538 publications

References 50 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

RNA transcription modulates phase transition-driven nuclear body assembly

The Activity-Dependent Regulation of Protein Kinase Stability by the Localization to P-Bodies

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