Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles

摘要

class="head no_bottom_margin" id="sec1title">IntroductionBiochemical reactions in the cell frequently have mutually exclusive solution requirements, leading to a need to keep them spatially separated. Membrane encapsulation is a commonly used strategy in roles ranging from controlling the flow of genetic information, via the nucleus and the ER, to maintaining the isolated acidic environment within a lysosome. An alternative strategy involves the formation of membraneless, proteinaceous organelles, including the prominent nucleolus (), PML bodies (), Cajal bodies () and nuclear speckles () in the nucleus, and P bodies and both stress and germ granules in the cytoplasm. These cellular structures have been described as coacervates () and are optically resolvable as spherical micron-sized droplets. The absence of a surrounding membrane enables these organelles to rapidly assemble or dissolve following changes in the cell’s environment and in response to intracellular signals, critical for cellular integrity and homeostasis () ().A striking feature of membraneless organelles is that their largely proteinaceous interior partially excludes the bulk aqueous phase (). Such organelles behave as liquid droplets. Fluorescence recovery after photobleaching (FRAP) experiments interrogating organelles such as the nucleolus and Cajal bodies indicate that their constituent molecules internally diffuse rapidly (), and P-granules, the worm analog of mammalian nuage or germ granules, condense from a pool of diffuse constituents following specific biological cues (). Moreover, spherical nucleoli of the amphibian oocyte have been observed to coalesce when in close contact and show a size distribution that obeys a simple power law, indicating the formation of liquid droplets (). On a residue level, sequences of low complexity, such as repeated RG, QN, and YG repeats, are important for forming RNA granules, stress granules and P bodies (). An understanding of the interactions that stabilize such structures and regulate their biogenesis, as well as a rationale for their biochemical function, has remained elusive.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 (). 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 (). Typical of non-membrane encapsulated organelles, nuages are generally spherical and dynamically change in number, size, and composition over their lifecycle (href="#bib38" rid="bib38" class=" bibr popnode">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 (href="#bib33" rid="bib33" class=" bibr popnode">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 (href="/pmc/articles/PMC4352761/figure/fig1/" target="figure" class="fig-table-link figpopup" rid-figpopup="fig1" rid-ob="ob-fig1" co-legend-rid="lgnd_fig1">Figures 1A and href="#app2" rid="app2" class=" sec">S2) (href="#bib22" rid="bib22" class=" bibr popnode">Forman-Kay and Mittag, 2013).href="/pmc/articles/PMC4352761/figure/fig1/" target="figure" rid-figpopup="fig1" rid-ob="ob-fig1">class="inline_block ts_canvas" href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=4352761_gr1.jpg" target="tileshopwindow">target="object" href="/pmc/articles/PMC4352761/figure/fig1/?report=objectonly">Open in a separate windowclass="figpopup" href="/pmc/articles/PMC4352761/figure/fig1/" target="figure" rid-figpopup="fig1" rid-ob="ob-fig1">Figure 1Ddx4 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. Ddx4^YFP organelles appear over time.(C) Differential interference contrast (DIC) and corresponding extended focus fluorescence intensity images of a HeLa cell expressing Ddx4^YFP. Ddx4^YFP 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 (href="#app2" rid="app2" class=" sec">Supplemental Experimental Procedures Section 5). The time is measured from the appearance of the first droplet.