Dynamics of the nuclear envelope during cell cycle in plants

Stereology of Allium cepa root meristem cells was done to evaluate changes in the nuclear envelope during cell cycle. A naturally synchronous population was labelled as binucleate by caffeine inhibition of cytokinesis. Growth of the nuclear envelope preferentially occurs from mid G2 to the next mid G1, most probably in relation to the reforming sister nuclei after mitosis. On the other hand, the number of nuclear pores doubles from mid G1 to mid G2, their growth rate being higher in the first half of interphase (from mid G1 to mid S). Hence, the new nuclear envelope probably lacks nuclear pores, which appear later.     

Redistribution of nuclear envelope associated antigen during the mitotic cycle.

Murine hybridomas were generated to DNA/tight binding proteins complex isolated from the residual nuclear structure following a procedure analogous to that yielding "empty" shells of nuclear envelope. A monoclonal antibody designated 2A8 was selected because of its differential immunostaining of mitotic cells of a synchronized mouse fibroblast cell culture L-929. The target antigen was rendered insoluble by a sequence of extractions of isolated nuclei of diverse cell types with detergents, urea, DNase I and alkali thus reproducing some solubility properties of proteins constituting an operationally defined residual nuclear matrix. The cognate polypeptide was localized on a subset of proteins of Mr 58-65 kDa, 70 kDa in isolated fibroblast nuclear matrices. The functional implication of the antigen in mitosis-related disassembly-assembly process of the nuclear matrix/envelope was detected. At prophase the antibody decorated the nuclear periphery and nuclear envelope fixed inward filaments. A fibrous network of cytoplasmic localization was stained in metaphase. At anaphase the antigen was dispositioned into peripheral fibrogranular clusters of polar orientation predominantly on one side of the nucleus. Proceeding to telophase a spreading fluorescence was manifested over the entire contour of the nuclear periphery to delineate the reforming nucleus. By immunogold electron microscopy of interphase cells the antigen was identified as evenly distributed in chromatin and interchromatin regions. At initiation of chromosome condensation in mitosis the label was detected predominantly in the chromosomal area.     

The permeability of the nuclear envelope in dividing and nondividing cell cultures

The objective of this study was to determine whether the permeability characteristics of the nuclear envelope vary during different phases of cellular activity. Both passive diffusion and signal-mediated transport across the envelope were analyzed during the HeLa cell cycle, and also in dividing, confluent (growth-arrested), and differentiated 3T3-L1 cultures. Colloidal gold stabilized with BSA was used to study diffusion, whereas transport was investigated using gold particles coated with nucleoplasmin, a karyophilic Xenopus oocyte protein. The gold tracers were microinjected into the cytoplasm, and subsequently localized within the cells by electron microscopy. The rates of diffusion in HeLa cells were greatest during the first and fifth hours after the onset of anaphase. These results correlate directly with the known rates of pore formation, suggesting that pores are more permeable during or just after reformation. Signal-mediated transport in HeLa cells occurs through channels that are located within the pore complexes and have functional diameters up to 230-250 A. Unlike diffusion, no significant differences in transport were observed during different phases of the cell cycle. A comparison of dividing and confluent 3T3-L1 cultures revealed highly significant differences in the transport of nucleoplasmin-gold across the envelope. The nuclei of dividing cells not only incorporated larger particles (230 A versus 190 A in diameter, including the protein coat), but the relative uptake of the tracer was about seven times greater than that in growth-arrested cells. Differentiation of confluent cells to adipocytes was accompanied by an increase in the maximum diameter of the transport channel to approximately 230 A.     

Increased nuclear envelope permeability and Pep4p-dependent degradation of nucleoporins during hydrogen peroxide-induced cell death

The death of yeast treated with hydrogen peroxide (H(2)O(2)) shares a number of morphological and biochemical features with mammalian apoptosis. In this study, we report that the permeability of yeast nuclear envelopes (NE) increased during H(2)O(2)-induced cell death. Similar phenomena have been observed during apoptosis in mammalian tissue culture cells. Increased NE permeability in yeast was temporally correlated with an increase in the production of reactive-oxygen species (ROS). Later, after ROS levels began to decline and viability was lost, specific nuclear pore complex (NPC) proteins (nucleoporins) were degraded. Although caspases are responsible for the degradation of mammalian nucleoporins during apoptosis, the deletion of the metacaspase gene YCA1 had no effect on the stability of yeast nucleoporins. Instead, Pep4p, a vacuolar cathepsin D homolog, was responsible for the proteolysis of nucleoporins. Coincident with nucleoporin degradation, a Pep4p-EGFP reporter migrated out of the vacuole in H(2)O(2)-treated cells. We conclude that increases in ROS and NPC permeability occur relatively early during H(2)O(2)-induced cell death. Later, Pep4p migrates out of vacuoles and degrades nucleoporins after the cells are effectively dead.     

The fate and origin of the nuclear envelope during and after mitosis in Amoeba proteus. I. Synthesis and behavior of phospholipids of the nuclear envelope during the cell life cycle

The synthesis and behavior of Amoeba proteus nuclear envelope (NE) phospholipids were studied. Most NE phospholipid synthesis occurs during G2 and little during mitosis or S. (A. proteus has no G1 phase). Autoradiographic observations after implantation of [3-H] choline nuclei into unlabeled cells reveal little turnover of NE phospholipid during interphase but during mitosis all the label is dispersed through the cytoplasm. Beginning at telophase all the label is dispersed through the cytoplasm. Beginning at telophase all the NE phospholipid label returns to the daughter NEs. This observation, along with the finding that no NE phospholipid synthesis occurs during mitosis or S, indicates that no de novo NE phospholipid production is required for newly forming NEs. Similarlyemetine, at concentrations that inhibit 97 percent of protein synthesis, does not prevent the post mitotic formation of NEs, suggesting that previously manufactured proteins are used in making new NEs. If a nucleus containing labeled NE phospholipids is transplanted into an unlabeled nucleate cell and the cell is allowed to grow and divide, the resultant four nuclei are equally labeled. This finding supports, but does not prove (see next paragraph), the conclusion that there probably is no continuity of the A. proteus NE during mitosis. When a phospholipid-labeled nucleus is implanted into a cell in mitosis, the grafted nucleus is not induced to enter mitosis. There is, however, a marked increase in the turnover of that nucleus's NE phospholipids with no apparent breakdown of the NE; this indicated that the mitotic cytoplasm possesses a factor that stimulates NE phospholipid exchange with the cytoplasm. That enhanced turnover is not accompanied by visible structural alteration makes less certain the earlier conclusion that no NE continuity exists during mitosis. Perhaps the most important finding in this study is that there are present, at restricted times in the cell cycle, factors capable of inducing accelerated exchange of structural components without microscopically detectable disruptions of structure.     

Regulation of proliferating cell nuclear antigen during the cell cycle

The proliferating cell nuclear antigen (PCNA), also known as cyclin and DNA polymerase delta auxiliary factor, is present in reduced amounts in nongrowing cells and is synthesized at a greater rate in the S phase of growing cells. The recently discovered involvement of PCNA in DNA replication suggested that this pattern of expression functions to regulate DNA synthesis. We have investigated this possibility further by examining the synthesis, stability, and accumulation of PCNA in HeLa cells fractionated by centrifugal elutriation into nearly synchronous populations of cells at various positions in the cell cycle. In these fractionated cells we found that there is an increase in the rate of PCNA synthesis with a peak in early S phase of the cell cycle, but the magnitude of the increase is only 2-3-fold. This change reflects similar changes in the amount of PCNA mRNA. The fluctuating synthesis of PCNA maintains this protein at a roughly constant proportion of the total cell protein, although the amount doubles/cell in the cell cycle. Consistent with this observation, the stability of PCNA does not differ significantly from that of total cellular protein in synchronized HeLa cells. We also observed that a maximum of one-third of the total PCNA is tightly associated with the nucleus, presumably in replication complexes, at the peak of S phase. We conclude that the cyclic synthesis of PCNA in cycling HeLa cells maintains PCNA in excess of the amount involved directly in DNA replication and the amount of the protein neither fluctuates significantly with the cell cycle nor is limiting for DNA synthesis.     

The redistribution of a conserved nuclear envelope protein during the cell cycle suggests a pathway for chromosome condensation

We describe a human autoantiserum that recognizes specific determinants present both on the nuclear envelope of interphase cells and the periphery of metaphase chromosomes. These determinants are highly conserved through evolution and present on a protein with an apparent molecular weight of 33,000. This 33 kd protein, which we call "perichromin," appears to be directly or indirectly bound to both interphase and metaphase DNA. Studies of the transformation of perichromin from a nuclear envelope association to a perichromosomal position during prophase suggests a pathway for chromosome organization throughout the cell cycle.     

Differential nuclear fluorescence during the cell cycle.

With a modified quinacrine dihydrochloride staining method interphase nuclei show differences in their fluorescent characteristics. In human embryonic fibroblasts and synchronized HeLa cells (S₃), it could be demonstrated that these patterns reflect their position within the cell cycle. This study provides further evidence for conformational changes of chromatin during interphase and offers a cytological method for distinguishing cell cycle stages.     

Dynamic remodeling of nuclear architecture during the cell cycle

The nuclear matrix is an integral part of nuclear structure which undergoes a profound reorganization during the cell cycle reflecting major changes in functional requirements. This includes the processes of DNA replication and gene expression at interphase and partitioning of the nuclear contents during mitosis. Using a monoclonal antibody (mAb2A) which specifically stains a novel nuclear meshwork which reorganizes during the cell cycle in Drosophila, we have initiated a study to: 1) more closely analyze this structural reorganization; 2) clone and characterize the antigens recognized by this antibody; and 3) isolate other interacting proteins in order to gain insight into the regulation of this process. The mAb2A-labeled structure changes from what appears as a diffuse meshwork at interphase to a distinct spindle-like scaffold at prophase. Since at metaphase the microtubules of the mitotic apparatus co-localize with the mAb2A spindle structure, a model is considered whereby the nuclear mAb2A-labeled scaffolding reorganizes during the cell cycle to provide a guide for the establishment of the mitotic apparatus. The mAb2A has identified two separate antigens, each of which shows similar distribution patterns. One of these antigens has been partially cloned and contains an unusual tandem set-thr kinase domain. The association of this kinase homologue with a nuclear scaffold which reorganizes during the cell cycle suggests that it may be involved in regulating changes in nuclear architecture during the cell cycle and/or in mediating the downstream consequences of such changes. © 1996 Wiley-Liss, Inc.     

Yeast nucleoporins involved in passive nuclear envelope permeability

The vertebrate nuclear pore complex (NPC) harbors an approximately 10-nm diameter diffusion channel that is large enough to admit 50-kD polypeptides. We have analyzed the permeability properties of the Saccharomyces cerevisiae nuclear envelope (NE) using import (NLS) and export (NES) signal-containing green fluorescent protein (GFP) reporters. Compared with wild-type, passive export rates of a classical karyopherin/importin (Kap) Kap60p/Kap95p-targeted NLS-GFP reporter (cNLS-GFP) were significantly faster in nup188-Delta and nup170-Delta cells. Similar results were obtained using two other NLS-GFP reporters, containing either the Kap104p-targeted Nab2p NLS (rgNLS) or the Kap121p-targeted Pho4p NLS (pNLS). Elevated levels of Hsp70 stimulated cNLS-GFP import, but had no effect on the import of rgNLS-GFP. Thus, the role of Hsp70 in NLS-directed import may be NLS- or targeting pathway-specific. Equilibrium sieving limits for the diffusion channel were assessed in vivo using NES-GFP reporters of 36-126 kD and were found to be greater than wild-type in nup188-Delta and nup170-Delta cells. We propose that Nup170p and Nup188p are involved in establishing the functional resting diameter of the NPC's central transport channel.     

The nuclear envelope in the plant cell cycle: structure, function and regulation

Background

Higher plants are, like animals, organisms in which successful completion of the cell cycle requires the breakdown and reformation of the nuclear envelope in a highly controlled manner. Interestingly, however, while the structures and processes appear similar, there are remarkable differences in protein composition and function between plants and animals.

Scope

Recent characterization of integral and associated components of the plant nuclear envelope has been instrumental in understanding its functions and behaviour. It is clear that protein interactions at the nuclear envelope are central to many processes in interphase and dividing cells and that the nuclear envelope has a key role in structural and regulatory events.

Conclusion

Dissecting the mechanisms of nuclear envelope breakdown and reformation in plants is necessary before a better understanding of the functions of nuclear envelope components during the cell cycle can be gained.     

Features of interactions of p68 anchorosomal protein with the nuclear envelope in the cell cycle

Chromatin associated with the nuclear envelope appears in the interphase nuclei as a layer of anchorosomes, granules 20-25 nm in diameter. The fraction of chromatin directly associated with the nuclear envelope is resistant to decondensation, shows a low level of DNA methylation, and contains specific acid-soluble proteins. However, mechanisms underlying the interaction of chromatin with the nuclear envelope are not fully understood. Specifically, it is not known whether anchorosomes are permanent structures or if they undergo reversible disassembly during mitosis, when contacts between chromatin and the nuclear envelope are destroyed. We obtained immune serum recognizing a 68 kDa protein from the nuclear envelopes fraction and studied the localization of this protein in interphase and mitotic cells. We show that this protein present in the NE/anchorosomal fraction does not remain bound with chromosomes during mitosis. It dissociates from chromosomes at the beginning of the prophase and then can be identified again at the periphery of the newly forming nuclei in the telophase.     

Cytoplasmic and nuclear protein kinases during the cell cycle.

Nuclear and cytoplasmic protein kinases were measured during the traverse of synchronous CHO cultures through G1 into S phase. Cells were synchronized by selective detachment of cells blocked in metaphase using colcemid. Nuclei were isolated and the protein kinases extracted from the nuclear preparation with 0.6 M NaCl. This procedure solubilized greater than 90% of the total protein kinase activity present in the nuclear preparation. DEAE chromatography of this extract showed 5 apparently different ionic forms of nuclear protein kinases. The nuclear protein kinases preferred casein and phosvitin to histone as substrates and were cyclic AMP-independent. Nuclear protein kinase activities increased greater than two-fold, when expressed as units of activity per cell nucleus, during G1 phase traverse, concomitant with a 70% increase in nuclear non-histone proteins (those soluble in 0.6 M NaCl). This resulted in only a 40% increase in the specific activities (units/microgram protein in 0.6 M NaCl extractable nuclear fraction) of these enzymes as cells progressed through G1 into S phase. This was in contrast to cytoplasmic cyclic AMP-dependent protein kinase activities which also increased two-fold during progression through G1 phase while total cellular protein increased less than 20%. Activation of, as well as synthesis of, cyclic AMP-dependent cytoplasmic protein kinases during G1 phase suggests a regulatory mechanism for precise temporal phosphorylation, whereas the constant specific activity in nuclear kinases during cell cycle is more compatible with the maintenance of bulk phosphorylation processes in the nucleus.     

Cellular and nuclear volume of human cells during the cell cycle

Summary The volumes of whole cells and nuclei of cultured human cells were studied at different times after synchronization of growth using the Coulter counter and scanning microphotometry. It was found that the increase in cell volume is compatible with both linear or exponential growth during the cell cycle. The growth of the nuclear volume is not correlated with the beginning of the DNA synthesis. The nuclear volume starts to increase already 6 h prior DNA synthesis. The data also indicate that the nuclear volume growth could proceed in two stages. The relation of this result to radiation sensitivity is discussed.This research was carried out under contract no. 215-76-10-BIO-D, Radiation Protection programme of the Commission of the European Community (Publication no. BIO 1747)     

Remodeling of nuclear architecture during the cell cycle in Drosophila embryos

Little is known about what determines the nuclear matrix or how its reorganization is regulated during mitosis. In this study we report on a monoclonal antibody, mAb2A, which identifies a novel nuclear structure in Drosophila embryos which forms a diffuse meshwork at interphase but which undergoes a striking reorganization into a spindle-like structure during pro- and metaphase. Double labelings with α-tubulin and mAb2A antibodies demonstrate that the microtubules of the mitotic apparatus co-localize with this mAb2A labeled structure during metaphase, suggesting it may serve a role in microtubule spindle assembly and/or function during nuclear division. That the mAb2A-labeled nuclear structure is essential for cell division and/or maintenance of nuclear integrity was directly demonstrated by microinjection of mAb2A into early syncytial embryos which resulted in a disintegration of nuclear morphology and perturbation of mitosis. © 1996 Wiley-Liss, Inc.     

Changes in envelope permeability during chloroplast development

Summary The permeability of the plastid envelope during the development of Avena sativa plastids was investigated by light scattering and uptake of various labelled compounds (malate, succinate, glutamate, -ketoglutarate, citrate, glycine, sucrose). The results presented show that a primary event during greening is a change in permeability, thereby allowing an increased transport of metabolites across the membranes of very early etio-chloroplast stages. The results are discussed in view of an adaption of the plastid envelope permeability to the changing requirements of externally synthesized precursors and intermediates during development.Abbreviations HEPES N-2-Hydroxyethylpiperazine-N-2-ethane-sulfonic acid - MES 2(N-Morpholino)ethane-sulfonic acid