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. 2010 Mar-Apr;1(2):202-11.
doi: 10.4161/nucl.1.2.11334. Epub 2010 Jan 28.

The traffic of proteins between nucleolar organizer regions and prenucleolar bodies governs the assembly of the nucleolus at exit of mitosis

Eleonora Muro  1 , Jeannine Gébrane-Younís, Aude Jobart-Malfait, Emilie Louvet, Pascal Roussel, Danièle Hernandez-Verdun
Affiliations

The traffic of proteins between nucleolar organizer regions and prenucleolar bodies governs the assembly of the nucleolus at exit of mitosis

Eleonora Muro et al. Nucleus. 2010 Mar-Apr.

Abstract

The building of nuclear bodies after mitosis is a coordinated event crucial for nuclear organization and function. The nucleolus is assembled during early G(1) phase. Here, two periods (early G1a and early G1b) have been defined. During these periods, the nucleolar compartments (DFC, GC) corresponding to different steps of ribosome biogenesis are progressively assembled. In telophase, rDNA transcription is first activated and PNBs (reservoirs of nucleolar processing proteins) are formed. The traffic of the processing proteins between incipient nucleoli and PNBs was analyzed using photoactivation. We demonstrate that the DFC protein fibrillarin passes from one incipient nucleolus to other nucleoli but not to PNBs, and that the GC proteins, B23/NPM and Nop52, shuttle between PNBs and incipient nucleoli. This difference in traffic suggests a way of regulating assembly first of DFC and then of GC. The time of residency of GC proteins is high in incipient nucleoli compared to interphase nuclei, it decreases in LMB-treated early G1a cells impairing the assembly of GC. Because the assembly of the nucleolus and that of the Cajal body at the exit from mitosis are both sensitive to CRM1 activity, we discuss the fact that assembly of GC and/or its interaction with DFC in early G1a depends on shuttling between PNBs and NORs in a manner dependent on Cajal body assembly.

Keywords: CRM1; Cajal body; EM structure; PNBs; cell cycle; living cell; nucleolus; photoactivation; traffic.

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Figures

Figure 1
Figure 1
Nucleolar assembly after telophase. (A) Time-lapse observation of living cells expressing GFP-Nop52. Thirty minutes after telophase Nop52 localizes in incipient nucleoli (large structures) and PNBs (small foci). After 90 minutes the incipient nucleoli are fused and the number of PNBs is low. (B) Number of PNBs evaluated in Z projections on time-lapse images. The presence of fibrillarin is limited in time (green), and the presence of Nop52 (black) is almost stable for nearly 1 hour and then decreases. X axis = time (min); Y axis = number of PNBs. (C) Schematic representation of nucleolar assembly based on time lapse observations. After telophase and before cytokinesis, 2 periods are observed in early G1 cells. Red: incipient nucleoli; black: PNBs. (D) Photoactivation of PAGFPB23/NPM in incipient nucleolus of an early G1 cell. The DsRed-Nop52 (red) shows one incipient nucleolus and several PNBs; PAGFP-B23/NPM (pre) is hardly visible. After activation (post 0 s), the PAGFP-B23/NPM is visible and then PNBs are labeled (10, 60, 120 seconds). (E) Z projection 2 min after activation showing the distribution of PAGFP-B23/NPM of one incipient nucleolus in all incipient nucleoli and PNBs.
Figure 2
Figure 2
Photoactivation of PAGFP-fibrillarin in incipient nucleolus of telophase and early-G1 cells. The DsRed-B23/NPM (c and d) and RFP-Nop56 (k and l) show incipient nucleoli and several PNBs (arrowheads); PAGFP-fibrillarin (pre) is hardly visible. 2 min after activation (post), the PAGFP-Fib is visible in activated incipient nucleoli and neighbouring incipient nucleoli (b, f and j) but not in PNBs (arrowheads). This is confirmed by the superimposition of green and red signals because only a red signal is visible in PNBs (d, h and l).
Figure 3
Figure 3
LMB blocks the cell cycle in early G1. (A) Phase contrast of unsynchronized HeLa cells treated for 2 hours with LMB. The number of early G1 cells (doublet linked by a cytoplasm bridge) is high. (B) Percentage of early G1 (black) depending on the duration of the LMB treatment compared to interphase (grey). The percentage increases with duration of treatment and can be maintained for up to 6 hours. (C) Cells blocked in early G1 after 120 minutes of LMB. In nuclei several large dark structures (phase contrast) containing B23 (GFP-B23 labeling) correspond to incipient nucleoli. In addition B23 is observed in many PNBs. The cytoplasmic bridge with the mid-body is visible in upper image. (D) Traffic of B23/NPM measured by photoactivation in early G1a for 2 minutes. Relative intensity in the activated incipient nucleoli, in control black curve and in LMB-treated cells (2 h) red curve (mean of 8 CTR and 10 LMB-treated cells). (E) Comparison of the t1/2 of B23/NPM traffic in incipient nucleoli of early G1 and interphase nucleoli in control (CTR) and LMB-treated (LMB) cells.
Figure 4
Figure 4
LMB blocks cells in early G1a. Cell cycle progression during LBM treatment observed by time lapse (GFP-B23/NPM cells). At 0 minutes, telophase (asterisk) cells are visible after 2 hours of LMB, and blocked doublet of early G1 cells is visible on the right side. After 10 minutes PNB-containing B23/NPM are visible but not the incipient nucleolus (arrow) that will be visible after 40 minutes. After 60 minutes the telophase cells are blocked in early G1a.
Figure 5
Figure 5
Progressive decrease of rDNA transcription under LMB. Two doublets of early G1a and one interphase (left) observed in phase contrast (phase). rDNA transcription (BrUTP) is detected in nucleoli of an interphase cell (Nu) and in incipient nucleoli indicated by arrows. GFP-B23/NPM is visible in the nucleolus (Nu), incipient nucleoli (arrows) and PNBs (small foci).
Figure 6
Figure 6
Ultrastructural organization of nucleoli undergoing 2 hours of LMB treatment. (A) Nucleolar organization is not modified during interphase. Fibrillar centers (asterisks), DFC (arrows) and granular component (GC). Bar: 1 µm. (B–C') Nucleolar organization is impaired in telophase. (B and C) correspond to 2 serial sections of the same nucleolus distant by 0.4 µm. The DFC is formed but with contrasted aggregates. (C') is the super-imposition of the DFC contour in black recognized by its dark and fibrillar texture, and in grey the GC without granule and loosely organized. Bar: 0.5 µm. (D) The GC is assembled in early G1 cells. Fibrillar centers (asterisks), DFC (arrows). Bar: 1 µm.
Figure 7
Figure 7
Knock-down of CRM1 reproduces the LMB effect. (A) In early G1 cells, LMB induces the localization of coilin in the cap (arrow) at the periphery of the incipient nucleoli. In control (CTR) cells, coilin (arrowhead) is localized in small foci. Bar: 5 µm. (B) Immunodetection of CRM1 as small nuclear foci already visible in early G1a nuclei. (C) Early G1 cells in which siRNAs against CRM1 were efficient (Δ CRM1): low CRM1 signal with antibodies compared to CRM1 positive cell in upper right corner (yellow arrow). Fibrillarin is in the cap (enlargment) at the periphery of the incipient nucleoli (white arrow); Nop52 is found both in incipient nucleoli and in PNBs (white arrow and enlargment).
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