Nucleolin modulates compartmentalization and dynamics of histone 2B-ECFP in the nucleolus

Abstract

Eukaryotic cells have 2 to ​3 discrete nucleoli required for ribosome synthesis. Nucleoli are phase separated nuclear sub-organelles. Here we examined the role of nuclear Lamins and nucleolar factors in modulating the compartmentalization and dynamics of histone 2B (H2B-ECFP) in the nucleolus. Live imaging and Fluorescence Recovery After Photobleaching (FRAP) of labelled H2B, showed that the depletion of Lamin B1, Fibrillarin (FBL) or Nucleostemin (GNL3), enhances H2B-ECFP mobility in the nucleolus. Furthermore, Nucleolin knockdown significantly decreases H2B-ECFP compartmentalization in the nucleolus, while H2B-ECFP residence and mobility in the nucleolus was prolonged upon Nucleolin overexpression. Co-expression of N-terminal and RNA binding domain (RBD) deletion mutants of Nucleolin or inhibiting 45S rRNA synthesis reduces the sequestration of H2B-ECFP in the nucleolus. Taken together, these studies reveal a crucial role of Nucleolin-rRNA complex in modulating the compartmentalization, stability and dynamics of H2B within the nucleolus.

Keywords: H2B; Nucleolus; lamin; nucleolin; nucleus; rRNA.

Figure 1.

Histone 2B-ECFP localizes in the nucleolus. (a) H2B-ECFP is distinctly localized in the nucleoplasm and the nucleolus. Top panel: nucleoplasmic localization of H2B-ECFP, Middle panel: localization of H2B-ECFP in the nucleolus (black arrowhead). Bottom panel: NLS-CFP localizes to the nucleoplasm and the nucleolus (black arrowhead). Scale bar ~5 μm. (b) All transfected cells show H2B-ECFP in the nucleoplasm, while ~40% of these cells harbor H2B-ECFP in the nucleolus. All cells show NLS-CFP in the nucleoplasm, while ~98% cells show NLS-CFP in the nucleolus, n = number of nuclei, data compiled from N = 2 independent biological replicates. (c) Immunostaining of Nucleolin marks nucleoli with H2B-ECFP in DLD1, HCT116 and MCF7 cells (white arrows). White outline demarcates single nucleus, scale bar ~5 μm. (d) Cells transfected with H2B-ECFP were immunostained with anti-histone 2B antibody and anti-nucleolin antibody to demarcate the nucleolus (white arrows), anti-histone 2B antibody detects both transfected and endogenous H2B in the nucleolus. (e) Independent knockdowns of Lamin A/C, B1, B2, FBL and GNL3 do not affect the extent of nucleolar localization of labeled H2B-ECFP, n = number of nuclei, data compiled from N = 3 independent biological replicates, error bars: SEM. Student’s t-test, p > 0.05 (n.s: not significant).

Figure 2.

Lamin B1 depletion enhances H2B–ECFP mobility in the nucleolus. (a–c) Western blots of whole cell lysates prepared from (a) LMNA/C (b) LMNB1 and (c) LMNB2 knockdown. Controls: Untreated, scramble siRNA. Loading control: Actin. (d–e) Representative images showing FRAP of H2B-ECFP in (d) nucleolus and (e) nucleus of control, LMNA/C Kd, LMNB1 Kd and LMNB2 Kd cells. Yellow box represents bleached ROI. Scale bar ~5 µm. (f–h) Normalized fluorescence recovery curves comparing recovery of H2B-ECFP in the nucleolus of control, (f) LMNA/C Kd (g) LMNB1 Kd and (h) LMNB2 Kd cells. (i) Relative mobile fractions of H2B-ECFP in the nucleolus as calculated from (f–h), showing increased mobility of H2B-ECFP in the nucleolus upon Lamin B1 Kd. (j–l) Normalized fluorescence recovery curves of H2B-ECFP in the nucleus of control, (j) LMNA/C Kd (k) LMNB1 Kd (l) LMNB2 Kd cells. (m) Relative mobile fractions of H2B-ECFP in the nucleus as calculated from (j–l). Lamin knockdown does not affect H2B-ECFP mobility in the nucleus, n = number of nuclei, data compiled from N = 3 independent biological replicates, error bars: SEM in recovery curves and bar graph. Student’s t-test, *p < 0.05.

Figure 3.

Depletion of Fibrillarin (FBL) and Nucleostemin (GNL3) enhances H2B–ECFP mobility in the nucleolus. (a, b) Western blots of whole cell lysates prepared from DLD1 cells upon knockdown of (a) Fibrillarin (FBL) (b) Nucleostemin (GNL3), Controls: untreated and respective scramble siRNA treated cells. Loading controls: Tubulin, Actin. (c, d) FRAP of H2B-ECFP in the (c) nucleolus and (d) nucleus of control, FBL and GNL3 Kd cells, respectively. Yellow box represents bleached ROI. Scale bar ~5 µm. (e, f) Normalized fluorescence recovery curves comparing recovery of H2B-ECFP in the nucleolus of control (e) FBL Kd and (f) GNL3 Kd cells. (g) Relative mobile fractions of H2B-ECFP in the nucleolus as calculated from (e, f). (h, i) Normalized fluorescence recovery curves comparing recovery of H2B-ECFP in the nucleus of control, (h) FBL Kd (i) GNL3 Kd cells. (j) Relative mobile fractions of H2B-ECFP in the nucleus as calculated from (h, i), n = number of nuclei, data from N = 3 independent biological replicates, error bars: SEM in recovery curves and bar graph. Student’s t-test, *p < 0.05, **p < 0.01.

Figure 4.

Nucleolin levels modulate compartmentation of H2B-ECFP in the nucleolus. (a) Representative images from live imaging of H2B-ECFP upon Nucleolin knockdown (NCL Kd) in DLD1 cells. Controls: untreated and scrambled siRNA treated cells. Scale bar ~5 µm. (b) (i) Western blots performed on whole cell lysates to detect Nucleolin levels in untreated, scramble and NCL siRNA treated DLD1 cells. Loading control: Actin. (ii) H2B-ECFP expression is marginally increased upon Nucleolin knockdown. Loading control: Tubulin. (c) Percent cells showing nucleolar H2B-ECFP compartments upon NCL Knockdown (Kd), n = number of nuclei, data from N = 3 independent biological replicates, error bars: SEM. Percent cells showing nucleolar H2B-ECFP upon NPM1 knockdown, n = number of nuclei, data from N = 2 independent biological replicates, error bars: SD. Percent cells showing nucleolar NLS-CFP upon NCL Kd, n = number of nuclei, data from N = 2 independent biological replicates, error bars: SD. Student’s t-test, ***p < 0.001. (d) Nucleolar H2B-ECFP upon NCL-GFP overexpression. DLD1 cells transfected with H2B-ECFP only (control, white bars) and co-transfected with H2B-ECFP and NCL-GFP (+NCL-GFP, black bars) imaged at intervals of 24, 48, and 72 h post transfection. Percent cells showing nucleolar H2B-ECFP upon 24 h of NPM1-GFP overexpression, n = number of nuclei, data from two independent biological replicates, N = 2, error bars: SD. (e) Representative images from live imaging H2B-ECFP transfected CRL1790, DLD1, HCT116 and MCF7 cells showing nucleolar localization of H2B-ECFP. Scale bar ~5 µm. (f) Western blots showing endogenous levels of NCL in CRL1790, DLD1, HCT116, and MCF7 cells. Loading controls: Tubulin, Actin. Intensity of Nucleolin normalized to loading control. (g) Extent of H2B-ECFP in the nucleolus in CRL1790, DLD1, HCT116, MCF7 cells and in DLD1 cells co-transfected with NCL-GFP, n = number of nuclei, data from two independent biological replicates, N = 2.

Figure 5.

Nucleolin levels positively correlate with H2B-ECFP mobility. (a) FRAP of H2B-ECFP in the nucleolus of DLD1, HCT116, MCF7 and DLD1 cells co-transfected with NCL-GFP (DLD1+ NCL-GFP OE), Scale bar ~5 µm. (b) Normalized fluorescence recovery curves of H2B-ECFP in the nucleolus. (c) Relative mobile fractions of H2B-ECFP as calculated from (b), n = number of nuclei, data from N = 3 independent biological replicates, error bars: SEM in recovery curves and bar graph. Student’s t-test, *p < 0.05.

Figure 6.

Nucleolin associates with H2B-ECFP in the nucleolus. (a) Co-immunoprecipitation (Co-IP) of endogenous Nucleolin and NCL-GFP with anti-Nucleolin antibody reveals its interaction with H2B-ECFP. Normal IgG used as a control for Co-IP. Anti-GFP antibody was used in western blot to detect H2B-ECFP. Data from N = 2 independent biological replicates. (b) Schematic showing three major domains of Nucleolin. The numbers denote the amino acid positions of the respective domains of Nucleolin. Representative images of DLD1 cells co-transfected with H2B-ECFP and Full length NCL (NCL FL), N-terminal deletion (NCLΔN), RNA binding domain deletion (NCLΔRBD) or GAR domain deletion (NCLΔGAR). H2B-ECFP and FL NCL show co-localization in the nucleolus. NCLΔRBD and NCLΔGAR show nucleoplasmic localization in addition to nucleolar localization. Scale bar, 5 µm. (c) Percent cells showing nucleolar H2B-ECP upon co-expression of NCL FL, NCLΔN, NCLΔRBD and NCLΔGAR, n = number of nuclei, data from N = 3 independent biological replicates, error bars: SEM. ANOVA, ***p < 0.001. (d) qRT-PCR for 45S pre-rRNA in vehicle (DMSO) and Actinomycin D (Act D, 0.05 μg/ml) treated cells, N = 2. 45S pre-rRNA level is downregulated in all cells upon Act D treatment. All statistics performed with respect to Control (+DMSO) cells, ANOVA, ***p < 0.001. (e) Representative images of cells expressing H2B-ECFP alone, or co-expressing NCL-GFP, upon DMSO or Act D treatment. Inset – Nucleolin speckles upon Act D treatment, do not show H2B-ECFP, Scale bar ~5 μm. (f) Quantification of percent cells showing nucleolar H2B-ECFP upon Act D treatment (from e), n = number of nuclei, N = 2 independent biological replicates. ANOVA, *p < 0.05.

Figure 7.

Speculative model of Nucleolin regulating nucleolar compartmentation and dynamics of H2B-ECFP. 1. Nucleolin interacts with H2B-ECFP via its N-terminal domain and shuttles it into the nucleolus. In the nucleolus, Nucleolin binds to pre-rRNA via its RNA binding domain and H2B-ECFP via its N-terminal domain, thus retaining H2B-ECFP in the nucleolus. It is likely that the relative rate of import of H2B-ECFP into the nucleolus is greater in the presence of Nucleolin. 2. In absence of the N-terminal domain, Nucleolin does not bind to H2B-ECFP, thereby reducing nucleolar pools of H2B-ECFP. 3. Nucleolin RBD deletion mutant binds to H2B-ECFP through its N-terminal domain and sequesters H2B-ECFP into the nucleolus. However, in the absence of RBD, H2B-ECFP is not retained in the nucleolus, as the RBD is required for binding to pre-rRNA. 4. GAR domain deletion mutant binds to H2B-ECFP and pre-rRNA and shows enhanced recruitment of H2B-ECFP into the nucleolus, similar to full length Nucleolin. 5. Nucleolin imports H2B-ECFP in the nucleolus but is unable to retain it in the nucleolus in the absence of pre-rRNA transcription inhibited by Act D.