Major oligomeric structural proteins of the HeLa nucleus

The three predominant polypeptides of the insoluble proteinaceous fraction from the HeLa cell nucleus polymerize in vitro upon oxidation of intrinsic sulfhydryl groups. The ease and specificity of this reaction indicate that these polypeptides exist as ordered oligomers in vivo. The comparable insoluble fraction from the rat liver nucleus also contains three predominant polypeptides of the same molecular weights, 65,000 71,000, and 75,000. The insoluble protein of the avian erythrocyte nuclear envelope consists principally of the 71,000- and the 75,000-dalton polypeptides. Indeed, in the avian erythrocyte nucleus these are the predominant polypeptides of the entire nucleus (Shelton, K., Cobbs, C., Povlishock, J. and Burkat, R., 1976, Arch. Biochem. Biophys.174, 177). Further, these avian polypeptides each form homogeneous covalently linked oligomers upon sulfhydryl oxidation (Cochran, D., Cobbs, C. and Shelton, K., 1977, J. Cell Biol.75, 151a). The insolubility, oligomeric disposition, and relative prominence of these polypeptides in a wide variety of cells indicate a fundamental structural role in the nucleus. Morphological features which may reflect this structural or skeletal role could be the nuclear envelope, the fibrous lamina, or perhaps an intrachromatinic matrix. The metabolism of the oligomeric polypeptides has been investigated in HeLa cells. Turnover of the HeLa insoluble nuclear protein is similar to that of the histones which are known to be stable proteins. The insoluble protein, including the oligomeric polypeptides, is synthesized in G1, S, and G2 phases of the cell cycle. This metabolic behavior indicates that the oligomeric polypeptides are reutilized in successive cell cycles and that synthesis accompanies nuclear and cellular expansion rather than deoxyribonucleohistone synthesis. This suggests that neither degradation nor selective synthesis of oligomeric polypeptides at a particular phase of the cell cycle are responsible for the breakdown and reformation of the interphase cell morphological features that occur during mitosis.     

Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression

TC4, a ras-like G protein, has been implicated in the feedback pathway linking the onset of mitosis to the completion of DNA replication. In this report we find distinct roles for TC4 in both nuclear assembly and cell cycle progression. Mutant and wild-type forms of TC4 were added to Xenopus egg extracts capable of assembling nuclei around chromatin templates in vitro. We found that a mutant TC4 protein defective in GTP binding (GDP-bound form) suppressed nuclear growth and prevented DNA replication. Nuclear transport under these conditions approximated normal levels. In a separate set of experiments using a cell-free extract of Xenopus eggs that cycles between S and M phases, the GDP- bound form of TC4 had dramatic effects, blocking entry into mitosis even in the complete absence of nuclei. The effect of this mutant TC4 protein on cell cycle progression is mediated by phosphorylation of p34cdc2 on tyrosine and threonine residues, negatively regulating cdc2 kinase activity. Therefore, we provide direct biochemical evidence for a role of TC4 in both maintaining nuclear structure and in the signaling pathways that regulate entry into mitosis.     

Cell-cycle-dependent, differential prenylation of proteins

Isoprenylated proteins related to cell growth have been detected during proliferation. Since cholesterogenesis (isoprenoid synthesis) is mandatory for cell proliferation, the observation of a temporally coordinated protein prenylation during the cell division cycle might constitute obligatory processes in the signalling pathway for initiating DNA replication and/or in maintaining the growing state. We have found such a definitive cell-cycle-phase-dependent pattern of prenylation for various classes of cytosolic and nuclear matrix proteins in synchronized HepG2 cells. Characteristic [3H]mevalonate incorporation began to increase during mid-to-late G1, just after cholesterol synthesis reached its apex, and peaked just prior to or coincident with mid S. Incorporation then declined subsequent to S (during G2) as cells approached mitosis. Prior to the rise in mevalonate incorporation into proteins, during early-to-mid G1, steady-state [14C]acetate incorporation into chromatographically resolved cholesterogenic lipid intermediates displayed a maximum only into cholesterol. However, during the late-G1/S interval, a singular peak of 14C incorporation was found for the farnesyl moiety (farnesol/nerolidol plus farnesyl diphosphate). Except for the farnesyl moiety, none of the other polyisoprenoids detected by our procedures showed any fluctuation in 14C incorporation subsequent to mid G1. These results support the proposal that subsequent to peak cholesterol synthesis in early-to-mid G1, the generation of a cholesterol-pathway-dependent set of post-translationally modified, polyisoprenylated proteins could constitute an obligatory step leading to the duplication of the cellular genome, thereby impelling transit through the cell cycle. The well known high flux through cholesterogenesis in tumors, which manifests an intrinsic lack of sensitivity to feedback inhibition and operates continuously, is consonant with this proposal.     

Localization of MPM-2 recognized phosphoproteins and tubulin during cell cycle progression in synchronized Vicia faba root meristem cells.

MPM-2 antibody reacts with a subset of mitotic phosphoproteins. We followed localization of MPM-2 immunoreactive material and localization of microtubules during cell cycle progression in a highly synchronous population of Vicia faba root meristem cells and isolated nuclei. The MPM-2 antibody labelling showed significant cell cycle dependence. MPM-2 nuclear reactivity was weak and homogeneous in G1 and S phase of the cell cycle and became stronger and heterogeneous during G2, resembling staining of the nuclear matrix, with maximum staining at the G2/M interface. Similarly the staining intensity of nucleoli increased from late G1 phase to nucleoli dispersion in early prophase. During mitosis MPM-2 immunoreactivity was associated with spindle configurations and the brightest signal was localized in kinetochores from prophase to metaphase.     

Mitotic architecture of the cell: the filament networks of the nucleus and cytoplasm

The skeletal framework of cells at the various stages of mitosis are prepared by extraction with nonionic detergent and examined by stereoscopic whole mount electron microscopy. The insoluble filament network remaining after the detergent-extraction and the depolymerization of microtubules is shown. The nonchromatin filament network of the nucleus, or nuclear matrix, becomes visible as the chromatin condenses at prophase. Filaments are associated with the chromosomes throughout mitosis. Parts of the chromosomes are associated with or are near the nuclear lamina at early stages. The nuclear lamina disappears at metaphase while chromosomes remain associated with filaments now continuous with the cytoplasmic network. Microtubules appear to be unnecessary for maintaining the chromosome position in these preparations since comparison of cells with and without microtubules shows no gross change in chromosome arrangement. The cellular filament network at metaphase and anaphase appears continuous from the plasma lamina to the chromosomes. The filament networks visualized here may be responsible for the prometaphase chromosome movement and participate in the formation of the midbody.     

Distribution of nuclear matrix proteins in interphase CHO cells and rearrangements during the cell cycle. An ultrastructural study

The nuclear matrix contains a group of residual non-histone proteins which remain structurally organized after extensive extraction of isolated nuclei with a high salt buffer, nucleases and a non-ionic detergent. Electron microscopic examination shows that the nuclear matrix is composed of a pore-complex lamina, an intranuclear network and residual nucleoli. In CHO cells biochemical analyses performed by one-dimensional SDS-PAGE show three major nuclear matrix polypeptides with molecular weights between 60 and 70 kDa. Polyclonal antibodies produced against these polypeptides were used to determine their nuclear distribution. Using immunoblotting, these proteins were found in whole nuclei, nuclear matrix, and in the intranuclear network but not in the pore-complex lamina. In order to determine the relationship between these structural proteins and the organization of the nucleus, the proteins were localized in situ. Ultrastructural detection was carried out by immunogold staining of thin sections of Lowicryl K4M-embedded cells. In interphase nuclei all condensed chromatin clumps were labelled. The nucleolus and the interchromatin granules were never immunogold-stained. During mitosis, the label was found to be associated with the chromosomes. This study shows that unlike the lamins, these 60-70 kDa nuclear matrix proteins are associated with the condensed chromatin throughout the cell cycle.     

Inactivation of 14-3-3sigma influences telomere behavior and ionizing radiation-induced chromosomal instability

Telomeres are complexes of repetitive DNA sequences and proteins constituting the ends of linear eukaryotic chromosomes. While these structures are thought to be associated with the nuclear matrix, they appear to be released from this matrix at the time when the cells exit from G(2) and enter M phase. Checkpoints maintain the order and fidelity of the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and cancer. The 14-3-3sigma gene has been reported to be a checkpoint control gene, since it promotes G(2) arrest following DNA damage. Here we demonstrate that inactivation of this gene influences genome integrity and cell survival. Analyses of chromosomes at metaphase showed frequent losses of telomeric repeat sequences, enhanced frequencies of chromosome end-to-end associations, and terminal nonreciprocal translocations in 14-3-3sigma(-/-) cells. These phenotypes correlated with a reduction in the amount of G-strand overhangs at the telomeres and an altered nuclear matrix association of telomeres in these cells. Since the p53-mediated G(1) checkpoint is operative in these cells, the chromosomal aberrations observed occurred preferentially in G(2) after irradiation with gamma rays, corroborating the role of the 14-3-3sigma protein in G(2)/M progression. The results also indicate that even in untreated cycling cells, occasional chromosomal breaks or telomere-telomere fusions trigger a G(2) checkpoint arrest followed by repair of these aberrant chromosome structures before entering M phase. Since 14-3-3sigma(-/-) cells are defective in maintaining G(2) arrest, they enter M phase without repair of the aberrant chromosome structures and undergo cell death during mitosis. Thus, our studies provide evidence for the correlation among a dysfunctional G(2)/M checkpoint control, genomic instability, and loss of telomeres in mammalian cells.     

Nuclear envelope dynamics.

The nuclear envelope (NE) provides a semi permeable barrier between the nucleus and cytoplasm and plays a central role in the regulation of macromolecular trafficking between these two compartments. In addition to this transport function, the NE is a key determinant of interphase nuclear architecture. Defects in NE proteins such as A-type lamins and the inner nuclear membrane protein, emerin, result in several human diseases that include cardiac and skeletal myopathies as well as lipodystrophy. Certain disease-linked A-type lamin defects cause profound changes in nuclear organization such as loss of peripheral heterochromatin and redistribution of other nuclear envelope components. While clearly essential in maintenance of nuclear integrity, the NE is a highly dynamic organelle. In interphase it is constantly remodeled to accommodate nuclear growth. During mitosis it must be completely dispersed so that the condensed chromosomes may gain access to the mitotic spindle. Upon completion of mitosis, dispersed NE components are reutilized in the assembly of nuclei within each daughter cell. These complex NE rearrangements are under precise temporal and spatial control and involve interactions with microtubules, chromatin, and a variety of cell-cycle regulatory molecules.     

Insect cells for the study of general problems in biology—Somatic inheritance

A central problem in biology concerns the mechanisms by which linear nucleic acid codes are translated into the 3-dimensional dynamic architecture of cells. The contents of cells are restricted in their movement by skeletons in their nuclei and cytoplasm. Every cell has an individual arrangement of skeletal components, which vary in time and space, while maintaining shape, internal order, and mechanical continuity with neighbouring cells. The precise pattern is realized by gene products that self-assemble at organizing centers oriented by delicate local stresses. However, the epidermis of caterpillars shows that other factors may contribute to skeletal patterns. The epidermis of caterpillars is composed of syncytial cell doublets formed by the retention of midbodies from mitosis to mitosis. It is an epithelium of Siamese twin cells. The interesting feature of these twins is that they have similar or even mirror image nuclear and cytoplasmic patterns. In most cells, sibling similarities are too short-lived to be noticed. In twin cells, the structural similarities are conserved, giving evidence for somatic inheritance, that is the survival and replication from one cell generation to the next of 3-dimensional arrangements that may not be completely specified by the nature and activity of the genetic material.     

Cell cycle-dependent alteration in NAC1 nuclear body dynamics and morphology

NAC1, a BTB/POZ family member, has been suggested to participate in maintaining the stemness of embryonic stem cells and has been implicated in the pathogenesis of human cancer. In ovarian cancer, NAC1 upregulation is associated with disease aggressiveness and with the development of chemoresistance. Like other BTB/POZ proteins, NAC1 forms discrete nuclear bodies in non-dividing cells. To investigate the biological role of NAC1 nuclear bodies, we characterized the expression dynamics of NAC1 nuclear bodies during different phases of the cell cycle. Fluorescence recovery after photobleaching assays revealed that NAC1 was rapidly exchanged between the nucleoplasm and NAC1 nuclear bodies in interphase cells. The number of NAC1 bodies significantly increased and their size decreased in the S phase as compared to the G?/G? and G? phases. NAC1 nuclear bodies disappeared and NAC1 became diffuse during mitosis. NAC1 nuclear bodies reappeared immediately after completion of mitosis. These results indicate that a cell cycle-dependent regulatory mechanism controls NAC1 body formation in the nucleus and suggest that NAC1 body dynamics are associated with mitosis or cytokinesis.     

Three-dimensional approaches to the residual structure of histone-depleted HeLa cell nuclei

Histone-depleted nuclei were prepared by high-salt extraction of interphase HeLa cell nuclei. A large amount of the nuclear DNA remained associated with a rapidly sedimenting residual nuclear structure including cytoplasmic (intermediate filament) and nuclear (matrix and lamina) proteins. Electron microscopy allowed detection in the insoluble structure of a residual nuclear envelope, nucleolar residues, and an intranuclear network whose correspondence with components of in situ fixed nuclei is discussed. Using three-dimensional electron microscopy, it is further demonstrated that the salt-insoluble structure remaining after histone depletion in 2 M NaCl is highly ordered. This is of the utmost importance when considering the roles reportedly ascribed to this structure in nuclear functions.     

Asynchronous nuclear division cycles in multinucleated cells

Synchronous mitosis is common in multinucleated cells. We analyzed a unique asynchronous nuclear division cycle in a multinucleated filamentous fungus, Ashbya gossypii. Nuclear pedigree analysis and observation of GFP-labeled spindle pole bodies demonstrated that neighboring nuclei in A. gossypii cells are in different cell cycle stages despite close physical proximity. Neighboring nuclei did not differ significantly in their patterns of cyclin protein localization such that both G1 and mitotic cyclins were present regardless of cell cycle stage, suggesting that the complete destruction of cyclins is not occurring in this system. Indeed, the expression of mitotic cyclin lacking NH(2)-terminal destruction box sequences did not block cell cycle progression. Cells lacking AgSic1p, a predicted cyclin-dependent kinase (CDK) inhibitor, however, showed aberrant multipolar spindles and fragmented nuclei that are indicative of flawed mitoses. We hypothesize that the continuous cytoplasm in these cells promoted the evolution of a nuclear division cycle in which CDK inhibitors primarily control CDK activity rather than oscillating mitotic cyclin proteins.     

Dynamics of Human Replication Protein A Subunit Distribution and Partitioning in the Cell Cycle

Human replication protein A (RPA) is a three-subunit protein complex involved in DNA replication, repair, and recombination. To gain insight into the dynamics of subunit assembly, we examined the subcellular distribution of RPA subunits (p70, p34, and p11) during the cell cycle. All three subunits colocalized in G1 and S phases, showing a diffuse nuclear distribution in G1 but a dot-like nuclear pattern in S phase. During S phase, the subunits showed a pattern reminiscent of the replication granules/factories described by others as sites of replication machinery. In metaphase, p70 preferentially associated with the spindle poles, p34 was found on chromosomes, and p11 remained in the cytoplasm. In telophase, p70 and p34 appeared in the forming daughter nuclei; p11 remained in the cytoplasm until G1. Among the three subunits only p34 was associated with the nuclear matrix and this association persisted throughout the cell cycle. We conclude that (i) RPA complex assembly is differentially regulated, (ii) the replication machinery may be anchored to the nuclear matrix, and (iii) RPA subunits partition during mitosis and sort into daughter nuclei by different routes.     

Stabilization and Localization of Xist RNA are Controlled by Separate Mechanisms and are Not Sufficient for X Inactivation

We examined the effect of cell cycle progression on various levels of chromosome organization in Drosophila. Using bromodeoxyuridine incorporation and DNA quantitation in combination with fluorescence in situ hybridization, we detected gross chromosomal movements in diploid interphase nuclei of larvae. At the onset of S-phase, an increased separation was seen between proximal and distal positions of a long chromsome arm. Progression through S-phase disrupted heterochromatic associations that have been correlated with gene silencing. Additionally, we have found that large-scale G1 nuclear architecture is continually dynamic. Nuclei display a Rabl configuration for only ∼2 h after mitosis, and with further progression of G1-phase can establish heterochromatic interactions between distal and proximal parts of the chromosome arm. We also find evidence that somatic pairing of homologous chromosomes is disrupted during S-phase more rapidly for a euchromatic than for a heterochromatic region. Such interphase chromosome movements suggest a possible mechanism that links gene regulation via nuclear positioning to the cell cycle: delayed maturation of heterochromatin during G1-phase delays establishment of a silent chromatin state.     

植物细胞中NuMA类似蛋白的分布及其在细胞周期中的变化

免疫荧光染色结果说明植物细胞核内含有与抗动物ＮｕＭＡ多抗呈阳性交叉反应的多肽。选择性抽提并结合免疫荧光染色结果说明这种多肽位于核基质纤维蛋白网络上。免疫印迹反应显示胡萝卜（ＤａｕｃｕｓｃａｒｏｔａＬ．）悬浮培养细胞核基质蛋白与抗动物ＮｕＭＡ蛋白多抗的阳性反应条带为７４ｋＤ和７６ｋＤ。有丝分裂各期免疫荧光染色的结果表明植物细胞中的ＮｕＭＡ类似蛋白在有丝分裂过程中呈现有规律的变化。结合选择性抽提的有丝分裂各期的免疫荧光染色的结果表明核基质在此过程中也发生明显变化。应用选择性抽提并结合ＤＧＤ包埋去包埋电镜技术对植物细胞间期及有丝分裂期核基质的形态结构进行了观察。结果显示胡萝卜悬浮培养细胞间期核内存在一个非染色质性的纤维蛋白网络体系,而在正处于分裂的细胞中则未观察到。以上结果说明ＮｕＭＡ类似蛋白是核基质的组分之一并与有丝分裂密切相关。     

LncRNAs of Saccharomyces cerevisiae bypass the cell cycle arrest imposed by ethanol stress

Ethanol alters many subsystems of Saccharomyces cerevisiae, including the cell cycle. Two ethanol-responsive lncRNAs in yeast interact with cell cycle proteins, and here, we investigated the role of these RNAs in cell cycle. Our network dynamic modeling showed that higher and lower ethanol-tolerant strains undergo cell cycle arrest in mitosis and G1 phases, respectively, during ethanol stress. The higher population rebound of the lower ethanol-tolerant phenotype after stress relief responds to the late phase arrest. We found that the lncRNA lnc9136 of SEY6210 (a lower ethanol-tolerant strain) induces cells to skip mitosis arrest. Simulating an overexpression of lnc9136 and analyzing CRISPR–Cas9 mutants lacking this lncRNA suggest that lnc9136 induces a regular cell cycle even under ethanol stress, indirectly regulating Swe1p and Clb1/2 by binding to Gin4p and Hsl1p. Notably, lnc10883 of BY4742 (a higher ethanol-tolerant strain) does not prevent G1 arrest in this strain under ethanol stress. However, lnc19883 circumvents DNA and spindle damage checkpoints, maintaining a functional cell cycle by interacting with Mec1p or Bub1p even in the presence of DNA/spindle damage. Overall, we present the first evidence of direct roles for lncRNAs in regulating yeast cell cycle proteins, the dynamics of this system in different ethanol-tolerant phenotypes, and a new yeast cell cycle model.     

Fresh WNT into the regulation of mitosis

Canonical Wnt signaling triggering β-catenin-dependent gene expression contributes to cell cycle progression, in particular at the G1/S transition. Recently, however, it became clear that the cell cycle can also feed back on Wnt signaling at the G2/M transition. This is illustrated by the fact that mitosis-specific cyclin-dependent kinases can phosphorylate the Wnt co-receptor LRP6 to prime the pathway for incoming Wnt signals when cells enter mitosis. In addition, there is accumulating evidence that various Wnt pathway components might exert additional, Wnt-independent functions that are important for proper regulation of mitosis. The importance of Wnt pathways during mitosis was most recently enforced by the discovery of Wnt signaling contributing to the stabilization of proteins other than β-catenin, specifically at G2/M and during mitosis. This Wnt-mediated stabilization of proteins, now referred to as Wnt/STOP, might on one hand contribute to maintaining a critical cell size required for cell division and, on the other hand, for the faithful execution of mitosis itself. In fact, most recently we have shown that Wnt/STOP is required for ensuring proper microtubule dynamics within mitotic spindles, which is pivotal for accurate chromosome segregation and for the maintenance of euploidy.