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Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles.

Nott TJ, Petsalaki E, Farber P, Jervis D, Fussner E, Plochowietz A, Craggs TD, Bazett-Jones DP, Pawson T, Forman-Kay JD, Baldwin AJ - Mol. Cell (2015)

Bottom Line: These bodies are stabilized by patterned electrostatic interactions that are highly sensitive to temperature, ionic strength, arginine methylation, and splicing.Moreover, the bodies provide an alternative solvent environment that can concentrate single-stranded DNA but largely exclude double-stranded DNA.We propose that phase separation of disordered proteins containing weakly interacting blocks is a general mechanism for forming regulated, membraneless organelles.

View Article: PubMed Central - PubMed

Affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada; Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3QZ, UK.

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Ddx4 Spontaneously Self-Assembles to Form Organelles in Live Cells(A) Evolutionary relationships between the disordered regions of Ddx4 homologs and their domain architectures. Disordered regions (green) and locations of DEAD-box helicase domains (brown) are indicated.(B) Schematic showing the DEAD-box helicase domain of Ddx4 replaced with YFP before being transfected into HeLa cells. Ddx4YFP organelles appear over time.(C) Differential interference contrast (DIC) and corresponding extended focus fluorescence intensity images of a HeLa cell expressing Ddx4YFP. Ddx4YFP forms dense, spherical organelles in the nucleus. Cells were stained with antibodies to visualize nucleoli, PML bodies, nuclear speckles, and Cajal bodies as indicated, revealing that Ddx4 organelles are entirely distinct from these other bodies.(D) The variation in total droplet volume with time is explained by the Avrami equation for nucleated growth (Supplemental Experimental Procedures Section 5). The time is measured from the appearance of the first droplet.
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fig1: Ddx4 Spontaneously Self-Assembles to Form Organelles in Live Cells(A) Evolutionary relationships between the disordered regions of Ddx4 homologs and their domain architectures. Disordered regions (green) and locations of DEAD-box helicase domains (brown) are indicated.(B) Schematic showing the DEAD-box helicase domain of Ddx4 replaced with YFP before being transfected into HeLa cells. Ddx4YFP organelles appear over time.(C) Differential interference contrast (DIC) and corresponding extended focus fluorescence intensity images of a HeLa cell expressing Ddx4YFP. Ddx4YFP forms dense, spherical organelles in the nucleus. Cells were stained with antibodies to visualize nucleoli, PML bodies, nuclear speckles, and Cajal bodies as indicated, revealing that Ddx4 organelles are entirely distinct from these other bodies.(D) The variation in total droplet volume with time is explained by the Avrami equation for nucleated growth (Supplemental Experimental Procedures Section 5). The time is measured from the appearance of the first droplet.

Mentions: To address these questions, we have studied a dominant protein constituent of a membraneless organelle as a model. Ddx4 proteins are essential for the assembly and maintenance of the related nuage in mammals, P-granules in worms, and pole plasm and polar granules in flies (Liang et al., 1994). This epigenetically crucial nuage/chromatoid body (CB) family of membraneless organelles hosts components of an RNAi pathway, guarding spermatocytes and spermatids against the deleterious activity of transposable elements (Kotaja and Sassone-Corsi, 2007). Typical of non-membrane encapsulated organelles, nuages are generally spherical and dynamically change in number, size, and composition over their lifecycle (Meikar et al., 2011), appearing first in the juxtanuclear cytoplasm of early spermatocytes, moving toward the base of the flagellum during spermatogenesis before finally dispersing. A primary constituent of nuage is Ddx4 (Kotaja et al., 2006). In addition to a central DEAD-box RNA helicase domain that uses ATP to unwind short RNA duplexes, Ddx4 has extended N and C termini that are predicted to be intrinsically disordered (Figures 1A and S2) (Forman-Kay and Mittag, 2013).


Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles.

Nott TJ, Petsalaki E, Farber P, Jervis D, Fussner E, Plochowietz A, Craggs TD, Bazett-Jones DP, Pawson T, Forman-Kay JD, Baldwin AJ - Mol. Cell (2015)

Ddx4 Spontaneously Self-Assembles to Form Organelles in Live Cells(A) Evolutionary relationships between the disordered regions of Ddx4 homologs and their domain architectures. Disordered regions (green) and locations of DEAD-box helicase domains (brown) are indicated.(B) Schematic showing the DEAD-box helicase domain of Ddx4 replaced with YFP before being transfected into HeLa cells. Ddx4YFP organelles appear over time.(C) Differential interference contrast (DIC) and corresponding extended focus fluorescence intensity images of a HeLa cell expressing Ddx4YFP. Ddx4YFP forms dense, spherical organelles in the nucleus. Cells were stained with antibodies to visualize nucleoli, PML bodies, nuclear speckles, and Cajal bodies as indicated, revealing that Ddx4 organelles are entirely distinct from these other bodies.(D) The variation in total droplet volume with time is explained by the Avrami equation for nucleated growth (Supplemental Experimental Procedures Section 5). The time is measured from the appearance of the first droplet.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352761&req=5

fig1: Ddx4 Spontaneously Self-Assembles to Form Organelles in Live Cells(A) Evolutionary relationships between the disordered regions of Ddx4 homologs and their domain architectures. Disordered regions (green) and locations of DEAD-box helicase domains (brown) are indicated.(B) Schematic showing the DEAD-box helicase domain of Ddx4 replaced with YFP before being transfected into HeLa cells. Ddx4YFP organelles appear over time.(C) Differential interference contrast (DIC) and corresponding extended focus fluorescence intensity images of a HeLa cell expressing Ddx4YFP. Ddx4YFP forms dense, spherical organelles in the nucleus. Cells were stained with antibodies to visualize nucleoli, PML bodies, nuclear speckles, and Cajal bodies as indicated, revealing that Ddx4 organelles are entirely distinct from these other bodies.(D) The variation in total droplet volume with time is explained by the Avrami equation for nucleated growth (Supplemental Experimental Procedures Section 5). The time is measured from the appearance of the first droplet.
Mentions: To address these questions, we have studied a dominant protein constituent of a membraneless organelle as a model. Ddx4 proteins are essential for the assembly and maintenance of the related nuage in mammals, P-granules in worms, and pole plasm and polar granules in flies (Liang et al., 1994). This epigenetically crucial nuage/chromatoid body (CB) family of membraneless organelles hosts components of an RNAi pathway, guarding spermatocytes and spermatids against the deleterious activity of transposable elements (Kotaja and Sassone-Corsi, 2007). Typical of non-membrane encapsulated organelles, nuages are generally spherical and dynamically change in number, size, and composition over their lifecycle (Meikar et al., 2011), appearing first in the juxtanuclear cytoplasm of early spermatocytes, moving toward the base of the flagellum during spermatogenesis before finally dispersing. A primary constituent of nuage is Ddx4 (Kotaja et al., 2006). In addition to a central DEAD-box RNA helicase domain that uses ATP to unwind short RNA duplexes, Ddx4 has extended N and C termini that are predicted to be intrinsically disordered (Figures 1A and S2) (Forman-Kay and Mittag, 2013).

Bottom Line: These bodies are stabilized by patterned electrostatic interactions that are highly sensitive to temperature, ionic strength, arginine methylation, and splicing.Moreover, the bodies provide an alternative solvent environment that can concentrate single-stranded DNA but largely exclude double-stranded DNA.We propose that phase separation of disordered proteins containing weakly interacting blocks is a general mechanism for forming regulated, membraneless organelles.

View Article: PubMed Central - PubMed

Affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada; Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3QZ, UK.

Show MeSH
Related in: MedlinePlus