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

Zhu, Lian; Richardson, Tiffany; Wacheul, Ludivine; Wei, Ming; Feric, Marina; Whitney, Gena; Lafontaine, Denis L. J.; Brangwynne, Clifford P.

doi:10.1073/pnas.1903870116

Cited by 70 publications

(66 citation statements)

References 55 publications

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“…In addition, it appears that molecular dynamics of NCL is regulated by HDACs upon acetyl-CoA depletion (S7D and S7E Fig). As the molecular dynamics in a condensate can be seen as an indicator for its material property, these observations imply alterations in the phase material property of the nucleolus, which is reported to be tightly linked to the nucleolar function [86]. It would be worth noting that the clustered lysine residues that we found as TMP195-sensitive acetylation sites were located in the N-terminal intrinsically disordered region (IDR) of NCL ( S7C Fig) [87].…”

Section: Discussionmentioning

confidence: 54%

Acetylation-mediated remodeling of the nucleolus regulates cellular acetyl-CoA responses

et al. 2020

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The metabolite acetyl-coenzyme A (acetyl-CoA) serves as an essential element for a wide range of cellular functions including adenosine triphosphate (ATP) production, lipid synthesis, and protein acetylation. Intracellular acetyl-CoA concentrations are associated with nutrient availability, but the mechanisms by which a cell responds to fluctuations in acetyl-CoA levels remain elusive. Here, we generate a cell system to selectively manipulate the nucleo-cytoplasmic levels of acetyl-CoA using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing and acetate supplementation of the culture media. Using this system and quantitative omics analyses, we demonstrate that acetyl-CoA depletion alters the integrity of the nucleolus, impairing ribosomal RNA synthesis and evoking the ribosomal protein-dependent activation of p53. This nucleolar remodeling appears to be mediated through the class IIa histone deacetylases (HDACs). Our findings highlight acetylation-mediated control of the nucleolus as an important hub linking acetyl-CoA fluctuations to cellular stress responses.

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Section: Discussionmentioning

confidence: 54%

Acetylation-mediated remodeling of the nucleolus regulates cellular acetyl-CoA responses

et al. 2020

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“…In the absence of bound NPM1-C, the ferritin core is strongly excluded from nucleoli, with a ΔG tr of approximately +1.4 kcal/mol ( Fig. S7), consistent with large noninteracting assemblies being excluded from the nucleolus and other condensates [22][23][24] . However, upon increasing the valence of the core with light activation, its partitioning into the nucleolus increases, implying a more negative transfer free energy.…”

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confidence: 68%

Composition dependent phase separation underlies directional flux through the nucleolus

Riback

Zhu

Ferrolino

et al. 2019

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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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“…Hence, the result may suggest that the redistribution of mass inside the nucleus is caused by the formation of nucleoli, thus reducing the mass density of nucleoplasm. This could be further investigated by measuring the mass density of nucleoplasm and nucleoli whose material properties can be modulated by an optogenetic gelation strategy (62). Recent studies have shown that the cell growth rate is sizedependent and exhibits exponential growth (63)(64)(65), and the size of nucleoli scales with that of cells while nuclei grow slower in the development of C. elegans embryo (66).…”

Section: Discussionmentioning

confidence: 99%

The relative densities of cell cytoplasm, nucleoplasm, and nucleoli are robustly conserved during cell cycle and drug perturbations

Guck

2020

Preprint

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The cell nucleus is a compartment in which essential processes such as gene transcription and DNA replication occur. While the large amount of chromatin confined in the finite nuclear space could install the picture of a particularly dense organelle surrounded by less dense cytoplasm, recent studies have begun to report the opposite. However, the generality of this newly emerging, opposite picture has so far not been tested. Here, we used combined optical diffraction tomography (ODT) and epi-fluorescence microscopy to systematically quantify the mass densities of cytoplasm, nucleoplasm, and nucleoli of human cell lines, challenged by various perturbations. We found that the nucleoplasm maintains a lower mass density than cytoplasm during cell cycle progression by scaling its volume to match the increase of dry mass during cell growth. At the same time, nucleoli exhibited a significantly higher mass density than the cytoplasm. Moreover, actin and microtubule depolymerization and changing chromatin condensation altered volume, shape, and dry mass of those compartments, while the relative distribution of mass densities was generally unchanged. Our findings suggest that the relative mass densities across membrane-bound and membraneless compartments are robustly conserved, likely by different as of yet unknown mechanisms, which hints at an underlying functional relevance. This surprising robustness of mass densities contributes to an increasing recognition of the importance of physico-chemical properties in determining cellular characteristics and compartments.

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

Cited by 70 publications

References 55 publications

Acetylation-mediated remodeling of the nucleolus regulates cellular acetyl-CoA responses

Acetylation-mediated remodeling of the nucleolus regulates cellular acetyl-CoA responses

Composition dependent phase separation underlies directional flux through the nucleolus

The relative densities of cell cytoplasm, nucleoplasm, and nucleoli are robustly conserved during cell cycle and drug perturbations

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