Compositional adaptability in NPM1-SURF6 scaffolding networks enabled by dynamic switching of phase separation mechanisms

Ferrolino, Mylene C.; Mitrea, Diana M.; Michael, J. Robert; Kriwacki, Richard W.

doi:10.1038/s41467-018-07530-1

Cited by 94 publications

(115 citation statements)

References 42 publications

(69 reference statements)

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“…Within the GC, NPM1 appears to form facsimiles of percolated droplets in complex ribosomal proteins with Arg-rich motifs (17,30). A minimalist system that mimics the phase behavior of the GC comprises of a truncated version of NPM1, referred to as N130, and an Arg-rich peptide, designated as rpL5 (31)(32)(33). Both NPM1 and N130 form symmetric pentamers in the presence of Arg-rich peptides (79).…”

Section: Fig 10mentioning

confidence: 99%

“…Recent studies have focused on uncovering the defining features of scaffold proteins (13,15,(17)(18)(19)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) and RNA molecules (41)(42)(43). These studies suggest that scaffolds are biological instantiations of associative polymers (44) characterized by a so-called stickers-andspacers architecture (45).…”

Section: Introductionmentioning

confidence: 99%

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LASSI: A lattice model for simulating phase transitions of multivalent proteins

Choi

Dar

Pappu

2019

Preprint

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§ These authors contributed equally * pappu@wustl.edu are shown to be system-specific and allow us to rationalize experimental observations while also enabling the design of systems with bespoke phase behavior. LASSI can be deployed to study the phase behavior of multicomponent systems, which allows us to make direct contact with cellular biomolecular condensates that are in fact multicomponent systems.

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Section: Fig 10mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

LASSI: A lattice model for simulating phase transitions of multivalent proteins

Choi

Dar

Pappu

2019

Preprint

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“…Recombinant poly-histidine tagged full-length wild-type and C21T/C275T mutant NPM1 were expressed in BL21 (DE3) E. coli cells (Millipore Sigma, Burlington, MA) and purified as previously described 17 . Briefly, cells were lysed by sonication and proteins were isolated from the insoluble fraction using Ni-NTA affinity chromatography.…”

Section: Purification Of Non-ribosomal Proteinsmentioning

confidence: 99%

“…In addition to NPM1, key GC components include ribosomal RNA (rRNA), and multivalent poly-arginine motif-containing proteins (Arg-proteins) such as SURF6, and ribosomal proteins (rproteins). Utilizing a well-established system for GC phase separation in vitro 11,12,17,18 , we formed either NPM1-only droplets with 5% PEG as a crowder (Fig. 2D, bottom image) or multicomponent droplets containing NPM1, the N-terminus of SURF6 (SURF6N), and rRNA ( Fig.…”

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confidence: 99%

Composition dependent phase separation underlies directional flux through the nucleolus

Riback

Zhu

Ferrolino

et al. 2019

Preprint

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Intracellular bodies such as nucleoli, Cajal bodies, and various signaling assemblies, represent membraneless organelles, or condensates, that form via liquidliquid phase separation (LLPS) 1,2 . Biomolecular interactions, particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions (IDRs), are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems 3-6 have led to the concept that a single fixed saturation concentration (C sat ) is a defining feature of endogenous LLPS 7-9 , and has been suggested as a mechanism for intracellular concentration buffering 2,7,8,10 . However, the assumption of a fixed C sat remains largely untested within living cells, where the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed C sat . As the concentration of individual components is varied, their partition coefficients change, in a manner that can be used to extract thermodynamic interaction energies, that we interpret within a framework we term polyphasic interaction thermodynamic analysis (PITA). We find that heterotypic interactions between protein and RNA components stabilize a variety of archetypal intracellular condensates, including the nucleolus, Cajal bodies, stress granules, and P bodies. These findings imply that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA processing condensates such as the nucleolus, this stoichiometric self-tuning manifests in selective exclusion of fully-assembled RNP complexes, providing a thermodynamic basis for vectorial ribosomal RNA (rRNA) flux out of the nucleolus. The PITA methodology is conceptually straightforward and readily implemented, and it can be broadly utilized to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deep understanding of the thermodynamics of multicomponent intracellular phase behavior and its interplay with nonequilibrium activity characteristic of endogenous condensates.To determine the thermodynamics of LLPS for intracellular condensates we first focused on the liquid granular component (GC) of nucleoli within HeLa cells, in particular on the protein Nucleophosmin (NPM1), which is known to be a key driver of nucleolar phase separation 11,12 . Under typical endogenous expression levels, we estimate NPM1 concentration in the nucleoplasm to be roughly C dil~4 μM; from simple binary phase separation models (i.e. Regular solution theory) 13 , this apparent saturation concentration, C sat , is expected to be fixed even under varied NPM1 expression levels (Fig. 1C, Supplemental Text). Consistent with previous studies 11 , ...

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“…NPM1 is a highly expressed nucleolar protein that is particularly enriched in the granular component (GC), the outer layer of the nucleolus that is associated with assembling ribosomal proteins together with processed rRNA into mature preribosomal particles. Purified NPM1 undergoes phase separation in vitro in an rRNAdependent manner (18)(19)(20). FBL is a nucleolar protein that is enriched in the dense fibrillar component (DFC) of the nucleolus.…”

mentioning

confidence: 99%

Controlling the material properties and rRNA processing function of the nucleolus using light

Zhu

Richardson

Wacheul

et al. 2019

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

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The nucleolus is a prominent nuclear condensate that plays a central role in ribosome biogenesis by facilitating the transcription and processing of nascent ribosomal RNA (rRNA). A number of studies have highlighted the active viscoelastic nature of the nucleolus, whose material properties and phase behavior are a consequence of underlying molecular interactions. However, the ways in which the material properties of the nucleolus impact its function in rRNA biogenesis are not understood. Here we utilize the Cry2olig optogenetic system to modulate the viscoelastic properties of the nucleolus. We show that above a threshold concentration of Cry2olig protein, the nucleolus can be gelled into a tightly linked, low mobility meshwork. Gelled nucleoli no longer coalesce and relax into spheres but nonetheless permit continued internal molecular mobility of small proteins. These changes in nucleolar material properties manifest in specific alterations in rRNA processing steps, including a buildup of larger rRNA precursors and a depletion of smaller rRNA precursors. We propose that the flux of processed rRNA may be actively tuned by the cell through modulating nucleolar material properties, which suggests the potential of materials-based approaches for therapeutic intervention in ribosomopathies.

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Compositional adaptability in NPM1-SURF6 scaffolding networks enabled by dynamic switching of phase separation mechanisms

Cited by 94 publications

References 42 publications

LASSI: A lattice model for simulating phase transitions of multivalent proteins

LASSI: A lattice model for simulating phase transitions of multivalent proteins

Composition dependent phase separation underlies directional flux through the nucleolus

Controlling the material properties and rRNA processing function of the nucleolus using light

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