Article

Quantitative kinetic analysis of nucleolar breakdown and reassembly during mitosis in live human cells

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: 9.69). 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.

0 Followers
 · 
118 Views
  • Source
    [Show abstract] [Hide abstract]
    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 · 5.33 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We analyzed the behavior of the nucleolus, nucleolar structures and nucleolus organizer regions (NORs) during meiotic division in four species of phyllostomid bats that have different numbers and locations of NORs. Nucleoli began disassembly at leptotene, and the subcomponents released from the nucleolus were dispersed in the nucleoplasm, associated with perichromosomal regions, or they remained associated with NORs throughout division. In Phyllostomus discolor, a delay in nucleolus disassembly was observed; it disassembled by the end of pachytene. The RNA complexes identified by acridine orange staining were observed dispersed in the nucleoplasm and associated with perichromosomal regions. FISH with rDNA probe revealed the number of NORs of the species: one NOR in Carollia perspicillata, one pair in Platyrrhinus lineatus and P. discolor, and three pairs in Artibeus lituratus. During pachytene, there was a temporary dissociation of the homologous NORs, which returned to pairing at diplotene. The variation in the number (from one to three pairs) and location of NORs (in sex or autosomal chromosomes, at terminal or interstitial regions) did not seem to interfere with the nucleolar behavior of the different species because no variation in nucleolar behavior that could be correlated with the variation in the number and chromosomal location of NORs was detected.
    Genetics and molecular research: GMR 01/2011; 10(2):552-65. DOI:10.4238/vol10-2gmr1060 · 0.85 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.
    Molecular biology of the cell 11/2010; 21(21):3735-48. DOI:10.1091/mbc.E10-06-0508 · 5.98 Impact Factor