TIME SEQUENCE OF NUCLEAR PORE FORMATION IN PHYTOHEMAGGLUTININ-STIMULATED LYMPHOCYTES AND IN HELA CELLS DURING THE CELL CYCLE

The time sequence of nuclear pore frequency changes was determined for phytohemagglutinin (PHA)-stimulated human lymphocytes and for HeLa S-3 cells during the cell cycle. The number of nuclear pores/nucleus was calculated from the experimentally determined values of nuclear pores/µ² and the nuclear surface. In the lymphocyte system the number of pores/nucleus approximately doubles during the 48 hr after PHA stimulation. The increase in pore frequency is biphasic and the first increase seems to be related to an increase in the rate of protein synthesis. The second increase in pores/nucleus appears to be correlated with the onset of DNA synthesis. In the HeLa cell system, we could also observe a biphasic change in pore formation. Nuclear pores are formed at the highest rate during the first hour after mitosis. A second increase in the rate of pore formation corresponds in time with an increase in the rate of nuclear acidic protein synthesis shortly before S phase. The total number of nuclear pores in HeLa cells doubles from ~2000 in G₁ to ~4000 at the end of the cell cycle. The doubling of the nuclear volume and the number of nuclear pores might be correlated to the doubling of DNA content. Another correspondence with the nuclear pore number in S phase is found in the number of simultaneously replicating replication sites. This number may be fortuitous but leads to the rather speculative possibility that the nuclear pore might be the site of initiation and/or replication of DNA as well as the site of nucleocytoplasmic exchange. That is, the nuclear pore complex may have multiple functions.     

应用HeLa细胞染色体(簇)和蛙卵提取物进行细胞核体外重建试验

将HeLa细胞中期染色体(簇)、非洲爪蟾卵提取物和ATP再生体系混合温育,能够促使细胞核自发重建。在此非细胞体系中重建的细胞核处于一般细胞核大小范围,具有典型的双层核膜,核孔复合体、染色质、核纤层、核骨架等结构,核重建具有一个明显的过程;发现环形片层通过与核膜融合方式参与核膜和核孔复合体组装。     

Nuclear pores during the cell cycle in a slime mold,Physarum polycephalum

Freeze-fracture and thin sectioning techniques were used to follow in large synchronous plasmodia of Physarum polycephalum the changes in number and distribution of nuclear pores during the cell cycle. Using freeze-fracture, we determined that average pore frequency rises gradually from 14/μm² of nuclear envelope surface at early S to a value of about 22 just before prophase. Nuclear diameter averaged 3.3 γm at early S and increased to 4.3 μm at late G2. Calculating nuclear volume and average chromatin volume per nucleus with respect to time in the cell cycle leads to the conclusion that number of nuclear pores appears to be most directly related to amount of chromatin present per nucleus and to be independent of nuclear surface area.     

DNA content contributes to nuclear size control in Xenopus laevis

Cells adapt to drastic changes in genome quantity during evolution and cell division by adjusting the nuclear size to exert genomic functions. However, the mechanism by which DNA content within the nucleus contributes to controlling the nuclear size remains unclear. Here, we experimentally evaluated the effects of DNA content by utilizing cell-free Xenopus egg extracts and imaging of in vivo embryos. Upon manipulation of DNA content while maintaining cytoplasmic effects constant, both plateau size and expansion speed of the nucleus correlated highly with DNA content. We also found that nuclear expansion dynamics was altered when chromatin interaction with the nuclear envelope or chromatin condensation was manipulated while maintaining DNA content constant. Furthermore, excess membrane accumulated on the nuclear surface when the DNA content was low. These results clearly demonstrate that nuclear expansion is determined not only by cytoplasmic membrane supply but also by the physical properties of chromatin, including DNA quantity and chromatin structure within the nucleus, rather than the coding sequences themselves. In controlling the dynamics of nuclear expansion, we propose that chromatin interaction with the nuclear envelope plays a role in transmitting chromatin repulsion forces to the nuclear membrane.     

高等动物细胞核膜和核纤层结构、功能及动态变化调控机制

细胞核是真核细胞中最大的细胞器．高等动物细胞核主要由双层核膜、核孔复合体、核纤层、染色质和核仁等组成．在细胞有丝分裂期,细胞核呈现去装配和再装配等动态变化．在细胞分裂间期,核膜、核孔复合体和核纤层构成细胞核的外周结构,为遗传物质在染色质和核仁中的代谢提供了一个相对稳定的环境,同时调控细胞核内外的物质转运,在细胞增殖、分化、个体发育和细胞衰老等许多方面发挥着重要作用．本文主要对高等动物细胞核膜和核纤层结构、功能及动态变化调控机制等方面的研究进展进行简要综述．     

An upper limit of the ratio of DNA volume to nuclear volume exists in plants

The variations in nuclear DNA content from 2 x 10(2) to 2.5 x 10(5) Mbp are reported in higher plants. The major finding so far is that the genome size of plant species differs by three orders of magnitude, which are more variable than the other organisms. Investigations pertaining to the manner in which DNA is packaged in the nucleus provide us with basic information on the made of DNA existence in the plant nucleus. However, the fundamentals on nuclear DNA content and nuclear size, which underlie and enable the flexible containment of such large differences in nuclear DNA content, remain unknown. We analyzed the nuclear volumes of plants with 2C value DNA contents ranging from 3.2 x 10(2) to 1.0 x 10(5) Mbp. As a result, we obtained a constant ratio between the DNA volume and nuclear volume, which does not exceed 3%. Furthermore, we also demonstrate that the nuclear Rabl model of chromatin organisation is not a common 3-D structure, even in plants with large nuclear DNA contents. The existence of an upper limit of DNA volume ratio would present a basal parameter for the future insight into the nuclear organisation in higher plants.     

Innate structure of DNA foci restricts the mixing of DNA from different chromosome territories

The distribution of chromatin within the mammalian nucleus is constrained by its organization into chromosome territories (CTs). However, recent studies have suggested that promiscuous intra- and inter-chromosomal interactions play fundamental roles in regulating chromatin function and so might define the spatial integrity of CTs. In order to test the extent of DNA mixing between CTs, DNA foci of individual CTs were labeled in living cells following incorporation of Alexa-488 and Cy-3 conjugated replication precursor analogues during consecutive cell cycles. Uniquely labeled chromatin domains, resolved following random mitotic segregation, were visualized as discrete structures with defined borders. At the level of resolution analysed, evidence for mixing of chromatin from adjacent domains was only apparent within the surface volumes where neighboring CTs touched. However, while less than 1% of the nuclear volume represented domains of inter-chromosomal mixing, the dynamic plasticity of DNA foci within individual CTs allows continual transformation of CT structure so that different domains of chromatin mixing evolve over time. Notably, chromatin mixing at the boundaries of adjacent CTs had little impact on the innate structural properties of DNA foci. However, when TSA was used to alter the extent of histone acetylation changes in chromatin correlated with increased chromatin mixing. We propose that DNA foci maintain a structural integrity that restricts widespread mixing of DNA and discuss how the potential to dynamically remodel genome organization might alter during cell differentiation.     

Till disassembly do us part: a happy marriage of nuclear envelope and chromatin

A characteristic feature of eukaryotic cells is the presence of nuclear envelope (NE) which separates genomic DNA from cytoplasm. NE is composed of inner nuclear membrane (INM), which interacts with chromatin, and outer nuclear membrane, which is connected to endoplasmic reticulum. Nuclear pore complexes are inserted into NE to form transport channels between nucleus and cytoplasm. In metazoan cells, an intermediate filament-based meshwork called as nuclear lamina exists between INM and chromatin. Sophisticated collaboration of these molecular machineries is necessary for the structure and functions of NE. Recent research advances have revealed that NE dynamically communicates with chromatin and cytoskeleton to control multiple nuclear functions. In this mini review, I briefly summarize the basic concepts and current topics of functional relationships between NE and chromatin.     

Nuclear import of adenovirus DNA in vitro involves the nuclear protein import pathway and hsc70

Adenovirus, a respiratory virus with a double-stranded DNA genome, replicates in the nuclei of mammalian cells. We have developed a cytosol-dependent in vitro assay utilizing adenovirus nucleocapsids to examine the requirements for adenovirus docking to the nuclear pore complex and for DNA import into the nucleus. Our assay reveals that adenovirus DNA import is blocked by a competitive excess of classical protein nuclear localization sequences and other inhibitors of nuclear protein import and indicates that this process is dependent on hsc70. Previous work revealed that the hexon (coat) protein of adenovirus is the only major protein on the surface of the adenovirus nucleocapsid that docks at the nuclear pore complex. This, together with our finding that in vitro nuclear import of hexon is inhibited by an excess of classical nuclear localization sequences, suggests a role for the hexon protein in adenovirus DNA import. However, recombinant transport factors that are sufficient for hexon import in permeabilized cells do not support DNA import, indicating that there are other as yet unidentified factors required for this process.     

Noninvasive Monitoring of Apoptosis versus Necrosis in a Neuroblastoma Cell Line Expressing a Nuclear Pore Protein Tagged with the Green Fluorescent Protein

A fusion chimera between the integral nuclear pore membrane protein POM121 and GFP (green fluorescent protein) has been shown to correctly target to the nuclear pores when transiently expressed in a number of mammalian cell types. POM121-GFP is therefore an excellent marker for the noninvasive studies of the nuclear pores in living cells using fluorescence microscopy. We have established a line of neuroblastoma cells stably expressing the POM121-GFP fusion protein. We also monitored the nuclear envelope in living cells after induction of apoptosis or necrosis using 1 μM staurosporine or 100 μMp-benzoquinone, respectively. Interestingly, the POM121-GFP fluorescence was weaker or missing in the apoptotic cells. The disappearance of the nuclear pore marker accompanied apoptotic progression as judged by the degree of chromatin condensation and DNA fragmentation as analyzed by DNA staining and TUNEL assay, respectively. In contrast, the intensity of the nuclear rim fluorescence was unaffected in necrotic cells displaying an abnormal morphology with tilted nuclei. Thus, it was possible to distinguish between apoptotic and necrotic development in living cells using fluorescence microscopy. This cell line provides a fast and convenient model for screening suspected toxic xenobiotics.     

Sea urchin testicular cells evaluated by fluorescence microscopy of unfixed tissue

A procedure is presented for rapid, quantitative evaluation of cell and nuclear types present in the male gonad of the sea urchin. Vitally stained whole mounts of tissue fragments or dissociated cells are prepared, which reveal detailed 3-dimensional chromatin patterns and enough cytoplasmic features to provide reliable markers for most of the somatic and germ line cell types. Representative cellular morphologies are described. Nuclear volume changes during spermatogenesis are quantified. Spermatid nuclei contain an apparently interconnected network of heterochromatin. Regions relatively devoid of chromatin decrease in size as nuclear condensation proceeds and spherical nuclear shape is maintained. The major decrease in nuclear volume occurs prior to the late spermatid stage. The volume of the spermatozoan nucleus is achieved by the smallest late spermatid nucleus before the change from spherical to conoid morphology. The relationship of this morphological transition to sperm histone dephosphorylation is discussed.     

Studies on a novel DNA polymerase inhibitor group, synthetic sulfoquinovosylacylglycerols: inhibitory action on cell proliferation

Some chemically synthesized sulfoquinovosylmonoacylglycerols (SQMG)-sulfoquinovosyldiacylglycerols (SQDG) have been reported to selectively and strongly inhibit the activities of mammalian DNA polymerases alpha and beta in vitro. In this study, using human cancer cell lines, we investigated the effects of SQMG-SQDG on the DNA polymerase in the cells. In the presence of n-decane, the IC(50) values on cell growth were approximately 1-5 microM for SQMG and about 0.3-1 microM for SQDG. The values were almost the same as the in vitro enzyme inhibitory levels. The cell lines were arrested in early S-phase by SQMG-SQDG at the concentrations of 0.1-4.7 microM in a manner dependent on incubation time, suggesting that SQMG-SQDG blocked the primary step of DNA replication by inhibiting DNA polymerase, possibly alpha-type. We also demonstrated the localization of SQMG in the cell using the fluorescent SQMG analog, SQMGalpha-NBDD, which was synthesized in our laboratory. SQMGalpha-NBDD was localized in the nucleus and on the nuclear surface, but the binding site seemed not to be the DNA/chromatin, suggesting that the SQMG-SQDG might interact with molecules located close to the DNA/chromatin and on the nuclear surface. These results suggested a correlation between the in vitro biochemical action of the SQMG-SQDGs and their intracellular mode of action.     

Condensed chromatin and its underreplication during root differentiation in leguminosae

Interphase nuclear structure was studied in 15 leguminous species. Eleven species showed chromocentric interphase nuclei while the remaining 4 had reticulate nuclei. The number of chromocenters appeared to be dependent on the number of chromosomes (2n). The total proportion of condensed chromatin as determined by planimetry was found to vary from 11–24% in chromocentric nuclei and 29–62% in reticulate nuclei. The condensed chromatin amount showed a direct correlation with the nuclear DNA content (2C). Though the interphase nuclear structure remained same in differentiated cells, the amount of condensed chromatin was considerably less than that in the meristematic cells, indicating underreplication of heterochromatin during differentiation. HCl-Giemsa method seems to be the simplest method for detection of underreplication in plants.1. NCL Communication No. 35942. To whom all the correspondence should be addressed     

Chromatin organization in relation to the nuclear periphery

In the limited space of the nucleus, chromatin is organized in a dynamic and non-random manner. Three ways of chromatin organization are compaction, formation of loops and localization within the nucleus. To study chromatin localization it is most convenient to use the nuclear envelope as a fixed viewpoint. Peripheral chromatin has both been described as silent chromatin, interacting with the nuclear lamina, and active chromatin, interacting with nuclear pore proteins. Current data indicate that the nuclear envelope is a reader as well as a writer of chromatin state, and that its influence is not limited to the nuclear periphery.     

Ankyrin and band 3 differentially affect expression of membrane glycoproteins but are not required for erythroblast enucleation

During late stages of mammalian erythropoiesis the nucleus undergoes chromatin condensation, migration to the plasma membrane, and extrusion from the cytoplasm surrounded by a segment of plasma membrane. Since nuclear condensation occurs in all vertebrates, mammalian erythroid membrane and cytoskeleton proteins were implicated as playing important roles in mediating the movement and extrusion of the nucleus. Here we use erythroid ankyrin deficient and band 3 knockout mouse models to show that band 3, but not ankyrin, plays an important role in regulating the level of erythroid cell membrane proteins, as evidenced by decreased cell surface expression of glycophorin A in band 3 knockout mice. However, neither band 3 nor ankyrin are required for enucleation. These results demonstrate that mammalian erythroblast enucleation does not depend on the membrane integrity generated by the ankyrin-band 3 complex.     

Condensed chromatin in diploid and allopolyploid microseris species with different genome size: a quantitative electron microscopic study

Summary The species-specific proportion of chromatin in the condensed state was estimated by quantitative electron microscopic morphometry of nuclear sections in 9 diploid and 5 allopolyploid species of Microseris (Asteraceae). A positive correlation between the genome size (haploid DNA content, or C value) and the percentage of chromatin in the condensed state (as visible in ultrathin sections) was found in diploids (r=0.89). Nuclei of allopolyploid (tetraploid) species exhibit condensed chromatin in a percentage which corresponds to the average of the values found in the parents. This suggests that each parental genome controls chromatin condensation at interphase independently within the nucleus, and that the degree of condensation is not directly determined by the nuclear DNA content per se. Genome size differences among Microseris species may depend preferentially, but not entirely, on DNA fractions located in, and perhaps being the cause of, condensed chromatin.Dedicated to Professor F. Mechelke in honour of his 60^th birthday.