Organization of Microtubules during the Transition from Cytokinesis to Interphase in Protonemal Cells of Adiantum capillus-veneris L.

The reorganization of microtubules (MTs) from cytokinesis tointerphase was examined in protonemal cells of the fern Adiantumcapillus-veneris. During the reorganization, many MTs fannedout from the nuclear envelope towards the cell periphery. Newlyformed cortical MTs were located only near the nucleus and werearranged randomly. The randomly arranged cortical MTs were thenreplaced by an interphase array of cortical MTs that were orientedpredominantly parallel to the cell axis. At the boundary betweenthe new and the old cell wall, clusters of MTs were observedafter the formation of cortical MTs. Re-formation of MTs after depolymerization of MTs was also examined.Clusters of short MTs appeared only at the nuclear envelopewhen MTs had been depolymerized by exposure of cells to 100µM propyzamide at 0°C. Few MTs were formed at theboundary between the new and old cell walls. These results suggestthat, even in fern cells, the nuclear envelope might act asMT-organizing center during the establishment of the interphasearray of MTs. (Received June 21, 1995; Accepted January 23, 1996)     

A Predictive Computational Model of the Dynamic 3D Interphase Yeast Nucleus

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Highlights? We describe a predictive computational model of dynamic chromosomes in the yeast nucleus ? The model quantitatively recapitulates experimental data on nuclear organization ? The model predicts nuclear reorganization in response to treatment by rapamycin ? Large-scale nuclear organization is dominated by unspecific effects of crowded polymers     

Evolution of the Global Internal Dynamics of a Living Cell Nucleus during Interphase

Progress in cellular biology based on fluorescent microscopy techniques, shows that the spatial organization of the nucleus is dynamic. This dynamic is very complex and involves a multitude of phenomena that occur on very different time and size scales. Using an original light scattering experimental device, we investigated the global internal dynamics of the nucleus of a living cell according to the phases of the cell cycle. This dynamic presents two different and independent kinds of relaxation that are well separated in time and specific to the phase of the cell cycle.     

Spatial Distribution and Mobility of the Ran GTPase in Live Interphase Cells

The GTPase Ran is a key regulator of molecular transport through nuclear pore complex (NPC) channels. To analyze the role of Ran in its nuclear transport function, we used several quantitative fluorescence techniques to follow the distribution and dynamics of an enhanced yellow fluorescent protein (EYFP)-Ran in HeLa cells. The diffusion coefficient of the majority of EYFP-Ran molecules throughout the cells corresponded to an unbound state, revealing the remarkably dynamic Ran regulation. Although we observed no significant immobile Ran populations in cells, ∼10% of the cytoplasmic EYFP-Ran and 30% of the nuclear EYFP-Ran exhibited low mobility indicative of molecular interactions. The high fraction of slow nuclear EYFP-Ran reflects the expected numerous interactions of nuclear RanGTP with nuclear transport receptors. However, it is not high enough to support retention mechanisms as the main cause for the observed nuclear accumulation of Ran. The highest cellular concentration of EYFP-Ran was detected at the nuclear envelope, corresponding to ∼200 endogenous Ran molecules for each NPC, and exceeding the currently estimated NPC channel transport capacity. Together with the relatively long residence time of EYFP-Ran at the nuclear envelope (33 ± 14 ms), these observations suggest that only a fraction of the Ran concentrated at NPCs transits through NPC channels.     

Heritability and Control of Differentiated Function in Cultured Cells

An established tissue culture cell line which retains a differentiated function in vitro is described. The cell line is of connective tissue origin, and its characteristic property is the synthesis and secretion of acid mucopolysaccharides, mainly hyaluronic acid. This differentiated cell function, the activity of which depends on continuous gene action, was found to be possessed by each of eleven clonal substrains, and is therefore a genetically heritable cell character. Rate of acid mucopolysaccharide biosynthesis falls sharply under the influence of the environmental conditions existing in crowded cultures, and this rate also declines if protein synthesis is directly inhibited with puromycin. Environmental modification of a differentiated product of gene action is thus illustrated in this study.     

The Dynamic Organization of the Perinucleolar Compartment in the Cell Nucleus

The perinucleolar compartment (PNC) is a unique nuclear structure preferentially localized at the periphery of the nucleolus. Several small RNAs transcribed by RNA polymerase III (e.g., the Y RNAs, MRP RNA, and RNase P H1 RNA) and the polypyrimidine tract binding protein (PTB; hnRNP I) have thus far been identified in the PNC (Ghetti, A., S. PinolRoma, W.M. Michael, C. Morandi, and G. Dreyfuss. 1992. Nucleic Acids Res. 20:3671–3678; Matera, A.G., M.R. Frey, K. Margelot, and S.L. Wolin. 1995. J. Cell Biol. 129:1181–1193; Lee, B., A.G. Matera, D.C. Ward, and J. Craft. 1996. Proc. Natl. Acad. Sci. USA. 93: 11471–11476). In this report, we have further characterized this structure in both fixed and living cells. Detection of the PNC in a large number of human cancer and normal cells showed that PNCs are much more prevalent in cancer cells. Analysis through the cell cycle using immunolabeling with a monoclonal antibody, SH54, specifically recognizing PTB, demonstrated that the PNC dissociates at the beginning of mitosis and reforms at late telophase in the daughter nuclei. To visualize the PNC in living cells, a fusion protein between PTB and green fluorescent protein (GFP) was generated. Time lapse studies revealed that the size and shape of the PNC is dynamic over time. In addition, electron microscopic examination in optimally fixed cells revealed that the PNC is composed of multiple strands, each measuring ~80–180 nm diam. Some of the strands are in direct contact with the surface of the nucleolus. Furthermore, analysis of the sequence requirement for targeting PTB to the PNC using a series of deletion mutants of the GFP–PTB fusion protein showed that at least three RRMs at either the COOH or NH₂ terminus are required for the fusion protein to be targeted to the PNC. This finding suggests that RNA binding may be necessary for PTB to be localized in the PNC.     

Virion Assembly Factories in the Nucleus of Polyomavirus-Infected Cells

Most DNA viruses replicate in the cell nucleus, although the specific sites of virion assembly are as yet poorly defined. Electron microscopy on freeze-substituted, plastic-embedded sections of murine polyomavirus (PyV)-infected 3T3 mouse fibroblasts or mouse embryonic fibroblasts (MEFs) revealed tubular structures in the nucleus adjacent to clusters of assembled virions, with virions apparently “shed” or “budding” from their ends. Promyelocytic leukemia nuclear bodies (PML-NBs) have been suggested as possible sites for viral replication of polyomaviruses (BKV and SV40), herpes simplex virus (HSV), and adenovirus (Ad). Immunohistochemistry and FISH demonstrated co-localization of the viral T-antigen (Tag), PyV DNA, and the host DNA repair protein MRE11, adjacent to the PML-NBs. In PML^−/− MEFs the co-localization of MRE11, Tag, and PyV DNA remained unchanged, suggesting that the PML protein itself was not responsible for their association. Furthermore, PyV-infected PML^−/− MEFs and PML^−/− mice replicated wild-type levels of infectious virus. Therefore, although the PML protein may identify sites of PyV replication, neither the observed “virus factories” nor virus assembly were dependent on PML. The ultrastructure of the tubes suggests a new model for the encapsidation of small DNA viruses.     

Augmin Triggers Microtubule-Dependent Microtubule Nucleation in Interphase Plant Cells

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Thermodynamic Pathways to Genome Spatial Organization in the Cell Nucleus

The architecture of the eukaryotic genome is characterized by a high degree of spatial organization. Chromosomes occupy preferred territories correlated to their state of activity and, yet, displace their genes to interact with remote sites in complex patterns requiring the orchestration of a huge number of DNA loci and molecular regulators. Far from random, this organization serves crucial functional purposes, but its governing principles remain elusive. By computer simulations of a statistical mechanics model, we show how architectural patterns spontaneously arise from the physical interaction between soluble binding molecules and chromosomes via collective thermodynamics mechanisms. Chromosomes colocalize, loops and territories form, and find their relative positions as stable thermodynamic states. These are selected by thermodynamic switches, which are regulated by concentrations/affinity of soluble mediators and by number/location of their attachment sites along chromosomes. Our thermodynamic switch model of nuclear architecture, thus, explains on quantitative grounds how well-known cell strategies of upregulation of DNA binding proteins or modification of chromatin structure can dynamically shape the organization of the nucleus.     

A Polymer Model for the Structural Organization of Chromatin Loops and Minibands in Interphase Chromosomes

A quantitative model of interphase chromosome higher-order structure is presented based on the isochore model of the genome and results obtained in the field of copolymer research. G1 chromosomes are approximated in the model as multiblock copolymers of the 30-nm chromatin fiber, which alternately contain two types of 0.5- to 1-Mbp blocks (R and G minibands) differing in GC content and DNA-bound proteins. A G1 chromosome forms a single-chain string of loop clusters (micelles), with each loop ~1–2 Mbp in size. The number of ~20 loops per micelle was estimated from the dependence of geometrical versus genomic distances between two points on a G1 chromosome. The greater degree of chromatin extension in R versus G minibands and a difference in the replication time for these minibands (early S phase for R versus late S phase for G) are explained in this model as a result of the location of R minibands at micelle cores and G minibands at loop apices. The estimated number of micelles per nucleus is close to the observed number of replication clusters at the onset of S phase. A relationship between chromosomal and nuclear sizes for several types of higher eukaryotic cells (insects, plants, and mammals) is well described through the micelle structure of interphase chromosomes. For yeast cells, this relationship is described by a linear coil configuration of chromosomes.     

Nuclear Exclusion of Separase Prevents Cohesin Cleavage in Interphase Cells

During mitosis, equal transmission of the duplicated chromosomes demands a strict regulation of separase, which cleaves cohesin and triggers sister chromatid separation in anaphase. Vertebrate separase is inhibited by securin and the inhibitory phosphorylation of separase. However, knockout experiments indicate that securin is dispensable and the inhibitory phosphorylation was observed only in M phase cells. This begs the question how cohesin cleavage by separase is prevented in the absence these two mechanisms. Here we show that separase is excluded from cohesin by the nuclear envelope, which forms in telophase and disassembles in mitosis. The exclusion is achieved passively by its large physical mass and may be backed up by the CRM1-dependent nuclear export. A functional NES motif is identified in separase. We demonstrated that the nuclear envelope is sufficient to prevent active separase from cleaving nuclear cohesin. We propose that the nuclear exclusion is important to prevent cohesin cleavage during interphase in the absence of securin and the phosphorylation inhibition.     

Latent Enhancers Activated by Stimulation in Differentiated Cells

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Highlights? Latent enhancers are regulatory elements unmarked and unbound in differentiated cells ? Stimulus-dependent TFs co-opt lineage-determining TFs to activate latent enhancers ? Latent enhancers may confer short-term memory of environmental exposure ? Environmental stimulation qualitatively alters the pre-existing enhancer repertoire