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The nucleolus as a multiphase liquid condensate

Nature Reviews Molecular Cell Biology volume 22, pages 165–182 (2021)Cite this article

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Abstract

The nucleolus is the most prominent nuclear body and serves a fundamentally important biological role as a site of ribonucleoprotein particle assembly, primarily dedicated to ribosome biogenesis. Despite being one of the first intracellular structures visualized historically, the biophysical rules governing its assembly and function are only starting to become clear. Recent studies have provided increasing support for the concept that the nucleolus represents a multilayered biomolecular condensate, whose formation by liquid–liquid phase separation (LLPS) facilitates the initial steps of ribosome biogenesis and other functions. Here, we review these biophysical insights in the context of the molecular and cell biology of the nucleolus. We discuss how nucleolar function is linked to its organization as a multiphase condensate and how dysregulation of this organization could provide insights into still poorly understood aspects of nucleolus-associated diseases, including cancer, ribosomopathies and neurodegeneration as well as ageing. We suggest that the LLPS model provides the starting point for a unifying quantitative framework for the assembly, structural maintenance and function of the nucleolus, with implications for gene regulation and ribonucleoprotein particle assembly throughout the nucleus. The LLPS concept is also likely useful in designing new therapeutic strategies to target nucleolar dysfunction.

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Fig. 1: Nucleolar organization and its role in ribosome biogenesis.
Fig. 2: Support for the liquid-phase nature of nucleoli.
Fig. 3: Understanding nucleolar substructure and material properties.
Fig. 4: Nucleolar dynamics in mitosis.
Fig. 5: Dynamics of ribosome biogenesis in the nucleolus.
Fig. 6: Links between nucleolar structure and function.

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Acknowledgements

Research in the Lafontaine laboratory is supported by the Belgian Fonds de la Recherche Scientifique (F.R.S./FNRS) (‘RiboEurope’ European Joint Programme on Rare Diseases (EJP RD/JTC2019/PINT-MULTI) grant n°R.8015.19 and PDR grant n°T.0144.20), the Université Libre de Bruxelles (ULB), the Région Wallonne (SPW EER) (‘RIBOcancer’ FSO grant n°1810070), the Fonds Jean Brachet, the Internationale Brachet Stiftung and the Epitran COST action (CA16120). The Brangwynne laboratory is supported by the Howard Hughes Medical Institute, the St. Jude Research Collaborative on Membrane-less Organelles and the National Institutes of Health (NIH) (U01 DA040601).

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Authors and Affiliations

  1. RNA Molecular Biology, Fonds de la Recherche Scientifique (F.R.S./FNRS), Université Libre de Bruxelles Cancer Research Center (ULB-CRC), Center for Microscopy and Molecular Imaging (CMMI), Gosselies, Belgium

    Denis L. J. Lafontaine & Rümeyza Bascetin

  2. Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, USA

    Joshua A. Riback & Clifford P. Brangwynne

  3. HHMI, Princeton University, Princeton, NJ, USA

    Clifford P. Brangwynne

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  1. Denis L. J. Lafontaine
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  2. Joshua A. Riback
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  3. Rümeyza Bascetin
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  4. Clifford P. Brangwynne
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The authors contributed equally to all aspects of the article.

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Correspondence to Denis L. J. Lafontaine or Clifford P. Brangwynne.

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Nature Reviews Molecular Cell Biology thanks T. Pederson, R. Kriwacki and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Glossary

Protein trans-acting factors

Proteins important for ribosomal subunit biogenesis that interact only transiently with maturing ribosomal subunits; they are not found on mature ribosomes.

Small nucleolar RNAs

Antisense small RNAs involved in RNA modification, pre-rRNA folding and processing.

Phase behaviour

The collection of biophysical features exhibited by materials capable of undergoing phase transitions, including the conditions under which phase separation occurs and the interfacial properties (for example, surface tension) defining wetting and coarsening behaviour.

Liquid–liquid phase separation

(LLPS). The process by which a single, uniform liquid phase demixes into two compositionally distinct liquid phases. Oil and water mixtures are an everyday example of phase-separated mixtures.

Coacervate

A term often used in the chemistry field to describe a condensed phase arising from a type of polymeric phase transition.

Fluorescence recovery after photobleaching

(FRAP). A technique that utilizes intense laser illumination to irreversibly deactivate fluorescent molecules locally, after which the return of a fluorescence signal within that region is indicative of biomolecular exchange with non-bleached fluorescent molecules from the surrounding areas of the sample.

Ribosomal proteins

Proteins that are structurally integrated into the ribosome. The fully assembled human ribosome contains 80 ribosomal proteins, most of which are assembled together with the four ribosomal RNAs within the nucleolus, with a notable exception of the acidic proteins forming the P stalk, which are assembled in the cytoplasm.

Non-equilibrium

A system that is not within a global thermodynamic free-energy minimum. Although equilibrium approaches may be applicable in some cases, biological systems are fundamentally out of equilibrium, due to their continuous consumption of energy and production and degradation of molecules.

Surfactant

A ‘surface-acting’ molecule that tends to localize at the interface of two phases, reducing surface tension; amphiphilic molecules are classic surfactants, due to their ability to simultaneously interact with both water and relatively hydrophobic structures. Ki-67 protein has been proposed to act as a surfactant in the context of the perichromosomal region.

Perinucleolar chromatin

Condensed chromatin lining the nucleolus during interphase and enriched in specific genes to react to the environment and others (stress, sensory and so on).

Intrinsically disordered regions

(IDRs). Protein regions that exhibit considerable conformational heterogeneity. The biased amino acid sequences of IDRs encode an intrinsic preference for conformational disorder and an inability to fold into singular well-defined 3D structures under physiological conditions.

Partitioning

The preference of a component for one of two distinct phases, quantified as the ratio of concentrations in the two phases (defining the partition coefficient, for example K = Cnucleolus / Cnucleoplasm) or as the free energy of transfer (ΔGtransfer = –RTlog(K)).

P granule

A perinuclear condensate implicated in germ cell lineage maintenance in Caenorhabditis elegans. P granules may serve similar functions to polar granules or nuage, which regulate germ cell biology across animal cells.

Emulsions

Liquid–liquid phase-separated solutions in which droplets of one liquid phase are dispersed throughout another immiscible continuous liquid phase; for example, droplets of vinegar in oil.

Ostwald ripening

The process by which larger droplets grow at the expense of smaller droplets, as a result of droplet surface tension (Laplace pressure) causing a higher chemical potential within the smaller droplet.

Viscoelastic

A viscoelastic material is one whose response to applied stresses is intermediate between that of a purely elastic solid and that of a purely viscous liquid; striking but common examples include silly putty and corn starch/water mixtures.

Saturation concentration

The concentration of a structurally key biomolecule above which liquid–liquid phase separation occurs. Note that for multicomponent systems governed by heterotypic interactions, the saturation concentration may not be identical to the concentration outside the condensate.

Cajal bodies

(Also known as coiled bodies). Nuclear condensates containing coilin and survival of motor neuron protein (SMN). Cajal bodies are enriched in U snRNAs and share some protein components with the nucleolus, such as fibrillarin.

Stress granule

Cytoplasmic condensates that form in response to stress (for example, oxidative stress and heat stress).

P body

Cytoplasmic condensates involved in mRNA degradation.

Surface tension

A material parameter characterizing the energy per unit area associated with an interface between two distinct phases. Liquids tend to round up into spheres as spheres minimize the surface area and thus minimize the interfacial energy imposed by surface tension. As nucleoli exhibit multiple subphases, each interface between them will have its own surface tension.

Complex fluids

Typically soft, liquid-like materials that often contain multiple macromolecular components such as polymers or colloidal particles and that usually exhibit viscoelastic properties; an emulsion is a type of complex fluid.

Metastability

A physical system may be said to be metastable if it resides in a local thermodynamic minimum, which over longer periods may begin transitioning into a different state associated with an even lower (global) energy minimum.

Microrheology

A technique used to measure the rheological properties of a microscopic material. Passive microrheology consists of tracking Brownian motions/thermal fluctuations of probe particles embedded within soft materials to determine viscoelasticity. Active microrheology utilizes externally applied stresses (for example, through optical or magnetic tweezers) and is particularly attractive for studying non-equilibrium systems where fluctuating motion may not be purely thermal (for example, ATP-dependent fluctuations).

Nucleolar breakdown

The process of nucleolar disassembly at the onset of mitosis.

Nucleolar genesis

The process of nucleolar (re)assembly at the end of mitosis.

Spinodal decomposition

The process by which phase separation occurs spontaneously without any nucleation barriers, due to the negative curvature of the free-energy landscape.

Heterogeneous nucleation

A process by which nucleation and growth of one phase within another is facilitated by nucleation on a favourable pre-existing surface (such as nascent pre-ribosomal RNAs in the case of nucleolar genesis).

Precursor–product relationship

A relationship between A and B if the production of B depends upon the disappearance of A. This applies, for example, to RNA processing (between upstream and downstream intermediates) and to the successive intermediate condensates formed during nucleolar genesis.

Perichromosomal region

Liquid-like condensate made of nucleolar proteins forming a ‘sheath’ around the compacted mitotic chromosomes (absent at the centromeres).

Nucleolar-derived foci

Condensates consisting of nucleolar proteins and formed during mitosis after nuclear envelope breakdown.

Prenucleolar bodies

Nuclear condensates consisting of nucleolar protein formed during mitosis at the time of nuclear membrane reassembly.

Alu RNAs

RNAs encoded in Alu repeats, which are the most abundant repetitive genetic elements found in primates, representing up to 10% of the human genome.

RNA exosome

A 3′–5′ exoribonucleolytic and endonucleolytic multi-protein complex involved in RNA 3′ end formation, RNA turnover and RNA surveillance (quality control).

Nucleolar surveillance

A p53-dependent antitumoural surveillance pathway triggered upon ribosomal assembly dysfunction and involving the sequestration of Hdm2 by a complex consisting of ribosomal proteins uL5, uL18 and the 5S ribosomal RNA.

Central protuberance

A major architectural landmark of the large ribosomal subunit (60S) essential for ribosomal function; it promotes transmission of allosteric information between the functional centres of the large ribosomal subunit, and between the small and large subunits during translation.

Ribosomopathies

Congenital or somatic tissue-specific diseases resulting from mutations in ribosomal proteins or ribosome biogenesis factors and leading to the shortage of mature ribosomes and, generally, to a hypo-proliferation phenotype. The blood and the brain are prime targets of ribosomopathies. Ribosomopathies often lead to cancer owing to secondary mutations.

Amyotrophic lateral sclerosis

A disease that causes the death of neurons controlling voluntary muscles.

Frontotemporal dementia

Diseases that affect mostly the frontal and temporal lobes of the brain associated with personality, behaviour and language.

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Lafontaine, D.L.J., Riback, J.A., Bascetin, R. et al. The nucleolus as a multiphase liquid condensate. Nat Rev Mol Cell Biol 22, 165–182 (2021). https://doi.org/10.1038/s41580-020-0272-6

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