Quantitative kinetic analysis of nucleolar breakdown and reassembly during mitosis in live human cells. J Cell Biol

Division of Gene Regulation and Expression, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, Scotland, UK.
The Journal of Cell Biology (Impact Factor: 9.83). 10/2004; 166(6):787-800. DOI: 10.1083/jcb.200405013
Source: PubMed

ABSTRACT One of the great mysteries of the nucleolus surrounds its disappearance during mitosis and subsequent reassembly at late mitosis. Here, the relative dynamics of nucleolar disassembly and reformation were dissected using quantitative 4D microscopy with fluorescent protein-tagged proteins in human stable cell lines. The data provide a novel insight into the fates of the three distinct nucleolar subcompartments and their associated protein machineries in a single dividing cell. Before the onset of nuclear envelope (NE) breakdown, nucleolar disassembly started with the loss of RNA polymerase I subunits from the fibrillar centers. Dissociation of proteins from the other subcompartments occurred with faster kinetics but commenced later, coincident with the process of NE breakdown. The reformation pathway also follows a reproducible and defined temporal sequence but the order of reassembly is shown not to be dictated by the order in which individual nucleolar components reaccumulate within the nucleus after mitosis.

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    • "Strikingly, the r-chromatin association of RPA194 was completely lost in mitotic cells (Fig. 2B). This result was consistent with the previous observation that the pol I subunits are transiently released from NORs during pro-metaphase [13] [14].The enrichment of Nopp140, DKC1, and FBL at the rRNA coding region was also significantly decreased in mitosis (Fig. 2C). These results indicated that pol I and pre-rRNA processing factors were released from r-chromatin in mitosis. "
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    ABSTRACT: The nucleolus is the ribosome biogenesis center. The nucleolar structure is disrupted upon entry into mitosis and is formed in early G1 phase. To understand the molecular mechanisms of nucleolar assembly and disassembly,we have studiedthe mechanism of association between factors involved in pre-ribosome RNA (rRNA) processing and rRNA gene chromatin (r-chromatin). We found that the pre-rRNA transcription-processing linking factor Nopp140 and pre-rRNA processing factors such as DKC1 and fibrillarin (FBL) associate with r-chromatin during interphase, while Nopp140, DKC1, and FBL were released from r-chromatin in mitosis. The association of these factors with r-chromatin wasfound to be restored independentof pre-rRNA transcription in early G1 phase, buta mature nucleolar structure was not formed, suggesting that nucleolar assembly can be divided into at least two steps with respect topre-rRNA transcription. Moreover, we found that the r-chromatin association of Nopp140, DKC1, and FBLwas dependent on the transcription factor Upstream Binding Factor (UBF). However, we demonstrated that UBF alone was not sufficient to recruit these pre-rRNA processing factors to r-chromatin.Thus, UBF is necessary but not sufficient for the associations between pre-rRNA processing factors and r-chromatin.
    Biochemical and Biophysical Research Communications 11/2013; 443(1). DOI:10.1016/j.bbrc.2013.11.039 · 2.30 Impact Factor
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    • "The nucleolus is an experimental model on which many fundamental principles in cell biology were first discovered. It is one of the first cellular structures studied by modern live cell time-lapse microscopy, which reveals the precisely coordinated dismantling and re-assembly of this organelle during cell division [17]. Quantitative fluorescent microscopy also uncovers the dynamics of protein flux through the nucleolus, leading to the paradoxical principle of selforganisation in mammalian nuclei [18]. "
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    ABSTRACT: The nucleolus is the location of ribosomal biogenesis, and plays crucial regulatory roles in nuclear responses to stress. Here, we report a new and improved nucleolar isolation method, which is simpler and more efficient than the traditional method. The purity of nucleoli obtained by using the new protocol is comparable to that by using the classical method, as judged by electron microscopy, Western blotting and SILAC-based quantitative proteomics. Moreover, the improved efficiency of cell harvesting in the new method, biochemical events in the nucleolus could be "frozen" and captured at precisely controlled time points. Time-lapse nucleolar proteomics after serum stimulation in HeLa cell revealed for the first time that some nucleolar proteins respond to serum stimulation within a time period as short as the first 5min of serum re-stimulation. Proteins involved in ribosomal biogenesis and in DNA damage repair are among the most dynamic proteins during the first 10min after serum replenishment. Notably, the proliferation marker Ki-67 is also found to enter the nucleolus after serum replenishment. To our knowledge, this is the first study that demonstrates such fast responses in the nucleolus, further confirming the rapid plasticity of this organelle.
    Journal of proteomics 10/2012; 77. DOI:10.1016/j.jprot.2012.09.031 · 3.89 Impact Factor
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    • "Proteins required for the earliest steps of ribosome biogenesis, such as fibrillarin, leave PNBs first, while pre-ribosomal factors (e.g. Bop1, Nop52 or B23), which participate in subsequent steps, reside in PNBs for a longer period of time (Angelier et al., 2005; Dundr et al., 2000; Leung et al., 2004; Savino et al., 1999; Savino et al., 2001). Preribosomal factors in PNBs are rapidly exchanged with the rest of the nucleus and can establish functional interactions in PNBs, as shown for Nop52 and B23 (Muro et al., 2010). "
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    ABSTRACT: Understanding the relationship linking the topological dynamics of nuclear subdomains to their molecular function is a central issue in nucleus biology. Pre-nucleolar bodies (PNBs) are transient nuclear subdomains, which form at telophase and contain nucleolar proteins, snoRNPs and pre-ribosomal RNAs (pre-rRNAs). These structures gradually disappear in early G1 phase and are currently seen as reservoirs of nucleolar factors that participate to post-mitotic reassembly of the nucleolus. Here, we provide evidence from FISH and loss-of-function experiments in HeLa cells that PNBs are in fact active ribosome factories in which maturation of the pre-rRNAs transiting through mitosis resumes at telophase. We show that the pre-rRNA spacers are sequentially removed in PNBs when cells enter G1 phase, indicating regular pre-rRNA processing as in the nucleolus. Accordingly, blocking pre-rRNA maturation induces accumulation in PNBs of stalled pre-ribosomes characterized by specific pre-rRNAs and pre-ribosomal factors. The presence of pre-ribosomal particles in PNBs is corroborated by observation of these domains with correlative electron tomography. Most importantly, blocking pre-rRNA maturation also prevents the gradual disappearance of PNBs, which persist for several hours in the nucleoplasm. In a revised model, we propose that PNBs are autonomous extra-nucleolar ribosome maturation sites, whose orderly disassembly in G1 phase is driven by the maturation and release of their pre-ribosome content.
    Journal of Cell Science 07/2012; 125(19). DOI:10.1242/jcs.106419 · 5.43 Impact Factor
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