Lavering ED et al. (2022), Component analysis of nucleolar protein compart...

XB-ART-59133

Dev Growth Differ 2022 Aug 01;646:306-317. doi: 10.1111/dgd.12794.

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Component analysis of nucleolar protein compartments using Xenopus laevis oocytes.

Lavering ED , Petros IN , Weeks DL .

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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.

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Species referenced: Xenopus laevis
Genes referenced: elavl2 fbl gar1 gtpbp4 ncl nhp2 nop56 npm1 pak1ip1 pes1 rpl12 snu13

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	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 Î¼m
	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 37Â°C for 1 h (controls were left at 13Â°C) in OCM and then recovered at 13Â°C for 24â€‰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 (pâ€‰<â€‰.05). Scale bar = 10 Î¼m
	Figure 4. Nucleolar proteins after heat shock. Oocytes were heat shocked at 37Â°C for 1 h (controls were left at 13Â°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 Î¼m. (b) Graphical representation of a dataset of heat shock treatments showing the number of punctate spots per nucleolus from oocytes treated at 13Â°C (control) or 37Â°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 (pâ€‰<â€‰.05). Scale bar = 10 Î¼m
	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 Î¼m
	Supplementary Figure 1. Example plasmid map of Gar1 fluorescently fused with mCherry. Figure was generated using Snap Gene.
	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

References [+] :

Alberti, Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates. 2019, Pubmed