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The nucleolus is a multi-compartment, non-membrane-bound organelle within the nucleus. Nucleolar assembly is influenced by proteins capable of phase separation. Xenopus laevis oocytes contain hundreds of large nucleoli that provide experimental access for nucleoli that is unavailable in other systems. Here we detail methods to streamline the in vivo analysis of the compartmentalization of nucleolar proteins that are suspected of phase separation. The nucleolus is the main hub of ribosome biogenesis and here we present data supporting the division of proteins into nucleolar domains based on their function in ribosome biogenesis. We also describe the use of vital dyes such as Hoechst 33342 and Thioflavin T in nucleolar staining. Additionally, we quantify nucleolar morphology changes induced by heat shock and actinomycin D treatments. We suggest these approaches will be valuable in a variety of studies that seek to better understand the nucleolus, particularly those regarding phase separation. These approaches may also be instructive for other studies on phase separation, especially in the nucleus.
Figure 1. Fluorescent fusion protein nucleolar localization. (a) Schematic of fluorescent fusion protein expression within Xenopus laevis oocyte nucleoli, showing the expression of the canonical domain markers Npm1 (fused with RFP) and Fbl (fused with GFP). (b) Model of the localization of each of these proteins. *Canonical domain markers. (c) Images of representative nucleoli with the labeled fluorescent fusion expressed with either Npm1 or Fbl, the canonical domain markers for the granular component and the dense fibrillar component, respectively. Scale bar = 10 μm
Figure 2. Representative images of Hoechst 33342, Sybr Green II, Syto Green, and Thioflavin T nucleolar staining as indicated by the label on each image. The Hoechst images are whole field fluorescence images, while all other images are apotome section fluorescence images. Scale bar = 10 μm
Figure 3. Recovery of fibrillarin after heat shock. Oocytes were heat shocked at 37°C for 1 h (controls were left at 13°C) in OCM and then recovered at 13°C for 24 h in OCM. (a) Representative images of nucleoli expressing fluorescently labeled Npm1 and Fbl after heat shock treatment and recovery. All images are of unfixed samples. (b) Graphical representation of a heat shock and recovery treatment showing the number of punctate spots in the control vs. heat shock vs. recovery as represented by the dense fibrillar component protein fibrillarin (p < .05). Scale bar = 10 μm
Figure 4. Nucleolar proteins after heat shock. Oocytes were heat shocked at 37°C for 1 h (controls were left at 13°C) in OCM for 1 h. (a) Representative images of nucleoli expressing fluorescently labeled proteins after heat shock treatment. RFP-fused proteins are shown in the first column of, GFP-fused proteins are shown in the second column, the merged images are shown in the third column. All images are of unfixed samples. Scale bar = 10 μm. (b) Graphical representation of a dataset of heat shock treatments showing the number of punctate spots per nucleolus from oocytes treated at 13°C (control) or 37°C (heat shock) for 1 h as represented by the dense fibrillar component localization protein indicated in either red or green. PS = punctate spots, which are partial dense fibrillar components. Each experiment was repeated three times, all producing significant changes between untreated and heat shocked nucleoli (p < .05). Scale bar = 10 μm
Figure 5. (a) Fluorescence microscopy images of actinomycin D treatment of nucleoli from oocytes injected with Fbl-RFP and Npm1-GFP. (b) Quantification of the area occupied by Fbl and Npm1 before and after actinomycin D treatment. (c) Representative images of actinomycin D-treated Fbl-RFP/Gar1-GFP, Gar1-mCherry/Npm1-GFP, and Gtpbp4-mCherry/Fbl-GFP. Scale bar = 10 μm
Supplementary Figure 1. Example plasmid map of Gar1 fluorescently fused with mCherry. Figure was generated using Snap Gene.
Alberti,
Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates.
2019, Pubmed
Alberti,
Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates.
2019,
Pubmed
Andersen,
Nucleolar proteome dynamics.
2005,
Pubmed
Banani,
Compositional Control of Phase-Separated Cellular Bodies.
2016,
Pubmed
Bański,
Chaperones and multitasking proteins in the nucleolus: networking together for survival?
2010,
Pubmed
Berry,
Physical principles of intracellular organization via active and passive phase transitions.
2018,
Pubmed
Bienz,
The heat-shock response in Xenopus oocytes is controlled at the translational level.
1982,
Pubmed
,
Xenbase
Boeynaems,
Spontaneous driving forces give rise to protein-RNA condensates with coexisting phases and complex material properties.
2019,
Pubmed
Boisvert,
The multifunctional nucleolus.
2007,
Pubmed
Boke,
Amyloid-like Self-Assembly of a Cellular Compartment.
2016,
Pubmed
,
Xenbase
Bouche,
Unusual processing of nucleolar RNA synthesized during a heat shock in CHO cells.
1981,
Pubmed
Bouvet,
Nucleolin interacts with several ribosomal proteins through its RGG domain.
1998,
Pubmed
Brangwynne,
Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes.
2011,
Pubmed
,
Xenbase
Brown,
Specific gene amplification in oocytes. Oocyte nuclei contain extrachromosomal replicas of the genes for ribosomal RNA.
1968,
Pubmed
Carroll,
DNA recombination and repair in oocytes, eggs, and extracts.
1991,
Pubmed
,
Xenbase
Chen,
Nucleolar components involved in ribosome biogenesis cycle between the nucleolus and nucleoplasm in interphase cells.
2001,
Pubmed
Costantini,
Assessing the tendency of fluorescent proteins to oligomerize under physiologic conditions.
2012,
Pubmed
Decker,
P-bodies and stress granules: possible roles in the control of translation and mRNA degradation.
2012,
Pubmed
Dumont,
Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals.
1972,
Pubmed
,
Xenbase
Fassler,
Protein Aggregation and Disaggregation in Cells and Development.
2021,
Pubmed
Feric,
Coexisting Liquid Phases Underlie Nucleolar Subcompartments.
2016,
Pubmed
,
Xenbase
Frottin,
The nucleolus functions as a phase-separated protein quality control compartment.
2019,
Pubmed
Fuentes,
In vivo functional characterization of the Saccharomyces cerevisiae 60S biogenesis GTPase Nog1.
2007,
Pubmed
Gall,
Structure in the amphibian germinal vesicle.
2004,
Pubmed
,
Xenbase
Ginisty,
Nucleolin functions in the first step of ribosomal RNA processing.
1998,
Pubmed
,
Xenbase
Gurdon,
Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells.
1971,
Pubmed
,
Xenbase
Hamma,
The box H/ACA ribonucleoprotein complex: interplay of RNA and protein structures in post-transcriptional RNA modification.
2010,
Pubmed
Han,
Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies.
2012,
Pubmed
Hayes,
Dual roles for ATP in the regulation of phase separated protein aggregates in Xenopus oocyte nucleoli.
2018,
Pubmed
,
Xenbase
Hayes,
Amyloids assemble as part of recognizable structures during oogenesis in Xenopus.
2016,
Pubmed
,
Xenbase
Huang,
Protein NPM3 interacts with the multifunctional nucleolar protein B23/nucleophosmin and inhibits ribosome biogenesis.
2005,
Pubmed
Hyman,
Liquid-liquid phase separation in biology.
2014,
Pubmed
Imami,
Phosphorylation of the Ribosomal Protein RPL12/uL11 Affects Translation during Mitosis.
2018,
Pubmed
Iyer-Bierhoff,
SIRT7-Dependent Deacetylation of Fibrillarin Controls Histone H2A Methylation and rRNA Synthesis during the Cell Cycle.
2018,
Pubmed
Jumper,
Highly accurate protein structure prediction with AlphaFold.
2021,
Pubmed
Kar,
Quantitative nucleolar proteomics reveals nuclear re-organization during stress- induced senescence in mouse fibroblast.
2011,
Pubmed
Kato,
Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels.
2012,
Pubmed
Kato,
Cross-β Polymerization of Low Complexity Sequence Domains.
2017,
Pubmed
Kay,
Xenopus laevis: Practical uses in cell and molecular biology. Injections of oocytes and embryos.
1991,
Pubmed
,
Xenbase
Labhart,
Heat shock stabilizes highly unstable transcripts of the Xenopus ribosomal gene spacer.
1987,
Pubmed
,
Xenbase
Lam,
New insights into nucleolar structure and function.
2015,
Pubmed
Lambert,
FPbase: a community-editable fluorescent protein database.
2019,
Pubmed
Lin,
SeqNLS: nuclear localization signal prediction based on frequent pattern mining and linear motif scoring.
2013,
Pubmed
Lindström,
NPM1/B23: A Multifunctional Chaperone in Ribosome Biogenesis and Chromatin Remodeling.
2011,
Pubmed
Mais,
Molecular architecture of the amplified nucleoli of Xenopus oocytes.
2001,
Pubmed
,
Xenbase
Maji,
Functional amyloids as natural storage of peptide hormones in pituitary secretory granules.
2009,
Pubmed
McSwiggen,
Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences.
2019,
Pubmed
Paine,
Protein loss during nuclear isolation.
1983,
Pubmed
,
Xenbase
Pearl,
Development of Xenopus resource centers: the National Xenopus Resource and the European Xenopus Resource Center.
2012,
Pubmed
,
Xenbase
REYNOLDS,
NUCLEOLAR "CAPS" PRODUCED BY ACTINOMYCIN D.
1964,
Pubmed
Roger,
rDNA transcription during Xenopus laevis oogenesis.
2002,
Pubmed
,
Xenbase
Rohrmoser,
Interdependence of Pes1, Bop1, and WDR12 controls nucleolar localization and assembly of the PeBoW complex required for maturation of the 60S ribosomal subunit.
2007,
Pubmed
Scalenghe,
Large scale isolation of nuclei and nucleoli from vitellogenic oocytes of Xenopus laevis.
1978,
Pubmed
,
Xenbase
Scheer,
Effects of actinomycin D on the association of newly formed ribonucleoproteins with the cistrons of ribosomal RNA in Triturus oocytes.
1975,
Pubmed
Scott,
Characterization and prediction of protein nucleolar localization sequences.
2010,
Pubmed
Sloan,
The 5S RNP couples p53 homeostasis to ribosome biogenesis and nucleolar stress.
2013,
Pubmed
Smith,
Oogenesis and oocyte isolation.
1991,
Pubmed
,
Xenbase
Tchelidze,
Nucleolar sub-compartments in motion during rRNA synthesis inhibition: Contraction of nucleolar condensed chromatin and gathering of fibrillar centers are concomitant.
2017,
Pubmed
van Nues,
Box C/D snoRNP catalysed methylation is aided by additional pre-rRNA base-pairing.
2011,
Pubmed
VASSAR,
Fluorescent stains, with special reference to amyloid and connective tissues.
1959,
Pubmed
Wang,
Nucleolar Sequestration: Remodeling Nucleoli Into Amyloid Bodies.
2019,
Pubmed
Watkins,
The box C/D and H/ACA snoRNPs: key players in the modification, processing and the dynamic folding of ribosomal RNA.
2012,
Pubmed
Weber,
Inverse size scaling of the nucleolus by a concentration-dependent phase transition.
2015,
Pubmed
Wühr,
Deep proteomics of the Xenopus laevis egg using an mRNA-derived reference database.
2014,
Pubmed
,
Xenbase
Xu,
Thioflavin T as an efficient fluorescence sensor for selective recognition of RNA G-quadruplexes.
2016,
Pubmed
Xue,
PONDR-FIT: a meta-predictor of intrinsically disordered amino acids.
2010,
Pubmed
Zernicka-Goetz,
An indelible lineage marker for Xenopus using a mutated green fluorescent protein.
1996,
Pubmed
,
Xenbase
