BACKGROUND Living cells contain distinct subcompartments to facilitate spatiotemporal regulation of biological reactions. In addition to canonical membrane-bound organelles such as secretory vesicles and endoplasmic reticulum, there are many organelles that do not have an enclosing membrane yet remain coherent structures that can compartmentalize and concentrate specific sets of molecules. Examples include assemblies in the nucleus such as the nucleolus, Cajal bodies, and nuclear speckles and also cytoplasmic structures such as stress granules, P-bodies, and germ granules. These structures play diverse roles in various biological processes and are also increasingly implicated in protein aggregation diseases. ADVANCES A number of studies have shown that membrane-less assemblies exhibit remarkable liquid-like features. As with conventional liquids, they typically adopt round morphologies and coalesce into a single droplet upon contact with one another and also wet intracellular surfaces such as the nuclear envelope. Moreover, component molecules exhibit dynamic exchange with the surrounding nucleoplasm and cytoplasm. These findings together suggest that these structures represent liquid-phase condensates, which form via a biologically regulated (liquid-liquid) phase separation process. Liquid phase condensation increasingly appears to be a fundamental mechanism for organizing intracellular space. Consistent with this concept, several membrane-less organelles have been shown to exhibit a concentration threshold for assembly, a hallmark of phase separation. At the molecular level, weak, transient interactions between molecules with multivalent domains or intrinsically disordered regions (IDRs) are a driving force for phase separation. In cells, condensation of liquid-phase assemblies can be regulated by active processes, including transcription and various posttranslational modifications. The simplest physical picture of a homogeneous liquid phase is often not enough to capture the full complexity of intracellular condensates, which frequently exhibit heterogeneous multilayered structures with partially solid-like characters. However, recent studies have shown that multiple distinct liquid phases can coexist and give rise to richly structured droplet architectures determined by the relative liquid surface tensions. Moreover, solid-like phases can emerge from metastable liquid condensates via multiple routes of potentially both kinetic and thermodynamic origins, which has important implications for the role of intracellular liquids in protein aggregation pathologies. OUTLOOK The list of intracellular assemblies driven by liquid phase condensation is growing rapidly, but our understanding of their sequence-encoded biological function and dysfunction lags behind. Moreover, unlike equilibrium phases of nonliving matter, living cells are far from equilibrium, with intracellular condensates subject to various posttranslational regulation and other adenosine triphosphate–dependent biological activity. Efforts using in vitro reconstitution, combined with traditional cell biology approaches and quantitative biophysical tools, are required to elucidate how such nonequilibrium features of living cells control intracellular phase behavior. The functional consequences of forming liquid condensates are likely multifaceted and may include facilitated reaction, sequestration of specific factors, and organization of associated intracellular structures. Liquid phase condensation is particularly interesting in the nucleus, given the growing interest in the impact of nuclear phase behavior on the flow of genetic information; nuclear condensates range from micrometer-sized bodies such as the nucleolus to submicrometer structures such as transcriptional assemblies, all of which directly interact with and regulate the genome. Deepening our understanding of these intracellular states of matter not only will shed light on the basic biology of cellular organization but also may enable therapeutic intervention in protein aggregation disease by targeting intracellular phase behavior.

Liquid phase condensation in cell physiology and disease.

The SIR genes are determinants of life span in yeast mother cells. Here we show that life span regulation by the Sir proteins is independent of their role in nonhomologous end joining. The short life span of a sir3 or sir4 mutant is due to the simultaneous expression of a and a mating-type information, which indirectly causes an increase in rDNA recombination and likely increases the production of extrachromosomal rDNA circles. The short life span of a sir2 mutant also reveals a direct failure to repress recombination generated by the Fob1p-mediated replication block in the rDNA. Sir2p is a limiting component in promoting yeast longevity, and increasing the gene dosage extends the life span in wild-type cells. A possible role of the conserved SIR2 in mammalian aging is discussed.

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The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms

Size homeostasis in budding yeast requires that cells grow to a critical size before commitment to division in the late prereplicative growth phase of the cell cycle, an event termed Start. We determined cell size distributions for the complete set of approximately 6000 Saccharomyces cerevisiae gene deletion strains and identified approximately 500 abnormally small (whi) or large (lge) mutants. Genetic analysis revealed a complex network of newly found factors that govern critical cell size at Start, the most potent of which were Sfp1, Sch9, Cdh1, Prs3, and Whi5. Ribosome biogenesis is intimately linked to cell size through Sfp1, a transcription factor that controls the expression of at least 60 genes implicated in ribosome assembly. Cell growth and division appear to be coupled by multiple conserved mechanisms.

https://science.sciencemag.org/content/sci/297/5580/395.full.pdf

Systematic identification of pathways that couple cell growth and division in yeast.

The synthesis of ribosomes is one of the major metabolic pathways in all cells. In addition to around 75 individual ribosomal proteins and 4 ribosomal RNAs, synthesis of a functional eukaryotic ribosome requires a remarkable number of trans-acting factors. Here, we will discuss the recent, and often surprising, advances in our understanding of ribosome synthesis in the yeast Saccharomyces cerevisiae. These will underscore the unexpected complexity of eukaryotic ribosome synthesis.

Ribosome synthesis in Saccharomyces cerevisiae.

Nucleolin is an abundant protein of the nucleolus. Nucleolar proteins structurally related to nucleolin are found in organisms ranging from yeast to plants and mammals. The association of several structural domains in nucleolin allows the interaction of nucleolin with different proteins and RNA sequences. Nucleolin has been implicated in chromatin structure, rDNA transcription, rRNA maturation, ribosome assembly and nucleo-cytoplasmic transport. Studies of nucleolin over the last 25 years have revealed a fascinating role for nucleolin in ribosome biogenesis. The involvement of nucleolin at multiple steps of this biosynthetic pathway suggests that it could play a key role in this highly integrated process.

Structure and functions of nucleolin

The nucleolus: an organelle formed by the act of building a ribosome

We have identified a gene (NPI46) encoding a new prolyl cis-trans isomerase within the nucleolus of the yeast Saccharomyces cerevisiae. The protein encoded by NPI46 was originally found by us in a search for proteins that recognize nuclear localization sequences (NLSs) in vitro. Thus, NPI46 binds to affinity columns that contain a wild-type histone H2B NLS but not a mutant H2B NLS that is incompetent for nuclear localization in vivo. NPI46 has two domains, a highly charged NH2 terminus similar to two other mammalian nucleolar proteins, nucleolin and Nopp140, and a COOH terminus with 45% homology to a family of mammalian and yeast proline isomerases. NPI46 is capable of catalyzing the prolyl cis-trans isomerization of two small synthetic peptides, succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, as measured by a chymotrypsin-coupled spectrophotometric assay. By indirect immunofluorescence we have shown that NPI46 is a nucleolar protein. NPI46 is not essential for cell viability.

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Yeast NPI46 encodes a novel prolyl cis-trans isomerase that is located in the nucleolus

Several nucleolar proteins, such as nucleolin, NOP1/fibrillarin, SSB1, NSR1 and GAR1 share a common glycine and arginine rich structural motif called the GAR domain. To identify novel nucleolar proteins from fission yeast we screened Schizosaccharomyces pombe genomic DNA libraries with a probe encompassing the GAR structural motif. Here we report the identification and characterization of a S.pombe gene coding for a novel nucleolar protein, designated gar2. The structure of the fission yeast gar2 is reminiscent of that of nucleolin from vertebrates and NSR1 from Saccharomyces cerevisiae. In addition, like these proteins, gar2 has a nucleolar localisation. The disruption of the gar2+ gene affects normal cell growth, leads to an accumulation of 35S pre-rRNA and a decrease of mature 18S rRNA steady state levels. Moreover, ribosomal profiles of the mutant show an increase of free 60S ribosomal subunits and an absence of free 40S ribosomal subunits. gar2 is able to rescue a S.cerevisiae mutant lacking NSR1, thus establishing gar2 as a functional homolog of NSR1. We propose that gar2 helps the assembly of pre-ribosomal particles containing 18S rRNA.

Teri Mélèse

Papers

The nucleolus: an organelle formed by the act of building a ribosome

Yeast NPI46 encodes a novel prolyl cis-trans isomerase that is located in the nucleolus

gar2 is a nucleolar protein from Schizosaccharomyces pombe required for 18S rRNA and 40S ribosomal subunit accumulation

The amino terminus of mammalian nucleolin specifically recognizes SV40 T-antigen type nuclear localization sequences.

Nucleolar proteins that bind NLSs: a role in nuclear import or ribosome biogenesis?