Electron microscopic studies of giant nucleus-like structure formed by lambda DNA introduced into the cytoplasm of Xenopus laevis fertilized eggs and embryos

When bacteriophage lambda DNA was injected into the cytoplasm of the fertilized egg of Xenopus laevis, giant nucleus-like structures were assembled around the injected DNA. These nucleus-like structures survived during cleavage and were partitioned into blastomeres at the blastula stage. The nucleus-like structures formed in the uncleaved fertilized eggs and the blastula cells were both surrounded by a bilayer nuclear membrane with nuclear pore complexes. The ultrastructural features of the lambda DNA-induced nucleus-like structure were considerably different from those of the normal blastula nucleus: although the nuclear pore complexes appeared to be normal, the 'nucleoplasm' was much too homogeneous as compared with that of the normal nucleus.     

Formation of nucleus-like structure in the cytoplasm of lambda-DNA-injected fertilized eggs and its partition into blastomeres during early embryogenesis in Xenopus laevis

The fate of bacteriophage lambda-DNA was examined after injection into the fertilized eggs of Xenopus laevis. Injection of a large amount of lambda-DNA (ca. 24 ng) into a fertilized Xenopus egg induced the formation around the injected DNA of a giant nucleus-like structure which was surrounded by an apparently normal bilayered nuclear membrane with nuclear pore complexes. Southern blot analysis revealed the persistence of injected lambda-DNA until the blastula stage. The nucleus-like structure was partitioned into blastomeres during cleavage through a process of nuclear fission, and was maintained in a group of extraordinarily large blastomeres until the blastula stage.     

Assembly in vitro of nuclei active in nuclear protein transport: ATP is required for nucleoplasmin accumulation.

DNA (from bacteriophage lambda or Xenopus) is assembled into nucleus-like structures when mixed with an extract from Xenopus eggs. Electron microscopy shows that these in vitro-reconstituted nuclei possess complete double membranes; some, but not all, nuclei have pore complexes. Extracts depleted of their endogenous ATP (by addition of ATPases) cannot assemble nuclear envelopes visible by phase-contrast microscopy. Once synthetic nuclei are assembled, however, they are stable when ATP is subsequently depleted, although their chromatin becomes condensed. About one-fourth of the nuclei assembled in vitro from lambda DNA accumulate nuclear proteins such as nucleoplasmin. ATP depletion blocks nucleoplasmin accumulation both in vitro, in pre-assembled synthetic nuclei, and in vivo, in the nucleus of microinjected oocytes. However, nucleoplasmin previously accumulated by reconstituted nuclei or by the germinal vesicle in microinjected oocytes is retained after ATP depletion.     

Histone stoichiometry and DNA circularization in archaeal nucleosomes.

Recombinant (r)HMfB (archaealhistone B fromMethanothermusfervidus) formed complexes with increasing stability with DNA molecules increasing in length from 52 to 100 bp, but not with a 39 bp molecule. By using125I-labeled rHMfB-YY (an rHMfB variant with I31Y and M35Y replacements) and32P-labeled 100 bp DNA, these complexes, designated archaeal nucleosomes, have been shown to contain an archaeal histone tetramer. Consistent with DNA bending and wrapping, addition of DNA ligase to archaeal nucleosomes assembled with 88 and 128 bp DNAs resulted in covalently-closed monomeric circular DNAs which, following histone removal, were positively supercoiled based on their electrophoretic mobilities in the presence of ethidium bromide before and after relaxation by calf thymus topoisomerase I. Ligase addition to mixtures of rHMfB with 53 or 30 bp DNA molecules also resulted in circular DNAs but these were circular dimers and trimers. These short DNA molecules apparently had to be ligated into longer linear multimers for assembly into archaeal nucleosomes and ligation into circles. rHMfB assembled into archaeal nucleosomes at lower histone to DNA ratios with the supercoiled, circular ligation product than with the original 88 bp linear version of this molecule. Archaeal histones are most similar to the globular histone fold region of eukaryal histone H4, and the results reported are consistent with archaeal nucleosomes resembling the structure formed by eukaryal histone (H3+H4)2tetramers.     

Ultrastructural analysis of cytoplasmic intermediate filaments and the nuclear lamina in the mouse plasmacytoma cell line MPC-11 after the induction of vimentin synthesis

We examined cytoplasmic intermediate filaments (IFs) and the nuclear lamina in cells of the mouse plasmacytoma cell line MPC-11 (lacking both IF proteins and lamins A and C) after induction of vimentin synthesis with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) by means of whole-mount immunogold electron microscopy (IEM). The technique of IEM was modified to allow analysis of the cytoskeleton and nuclear lamina of cells grown in suspension culture employing antibodies against vimentin and lamin B. IEM showed that newly synthesized vimentin assembled into IFs which formed anastomosing networks throughout the cytoplasm, radiating primarily from the nucleus. The filaments decorated by gold-conjugated antibodies appeared to make contact with the lipid-depleted nuclear envelope residue either by directly terminating on it or through an indirect link via short fibers of varying diameter. Some filaments terminated on the subunits of the nuclear pore complexes but they did not pass through the pores. In the absence of lamins A and C, lamin B formed a nuclear lamina consisting of a globular-filamentous network anchoring the nuclear pore complexes.     

In vitro nuclear assembly with affinity-purified nuclear envelope precursor vesicle fractions, PV1 and PV2.

Nuclear envelope precursor vesicles were affinity purified from a Xenopus egg extract by a chromatin binding method. Vesicles bound to chromatin at 4 degrees C were dissociated with a high salt buffer and further fractionated into nuclear envelope precursor vesicle fractions 1 (PV1) and 2 (PV2) by differential centrifugation. PV1 contained larger vesicles. When chromatin was incubated in a Xenopus egg cytosol fraction supplemented with PV1, vesicles bound to chromatin, fused with each other, formed a bilayered nuclear envelope, and assembled into spherical small nuclei. However, the thus assembled nuclei did not grow to the normal size. Nuclear pore complexes were not found on the thus assembled nuclei. On the other hand, PV2 contained smaller vesicles. PV2 vesicles bound to chromatin, fused little with each other in the Xenopus egg cytosol fraction, and no nuclei were assembled. When PV1 supplemented with PV2 was used for the nuclear assembly reaction, the assembled nuclei grew to the normal size. Nuclear pore complexes existed in the thus assembled nuclear envelopes. These results suggested that 1) two vesicle populations, PV1 and PV2, are necessary for the assembly of normal sized nuclei, 2) PV1 contains a chromatin targeting molecule(s) and membrane fusion machinery, 3) PV2 contains a chromatin targeting molecule(s) and a molecule(s) necessary for nuclear pore complex assembly, and 4) PV1 has the ability to assemble a nuclear membrane, and PV2 is necessary for the assembly of nuclear pore complexes and for nuclei to grow to the normal size. An in vitro nuclear assembly system constituted with affinity-purified vesicle fractions, PV1 and PV2, was established.     

Nuclear formation in a Drosophila cell-free system

A cell-free preparation obtained from 0- to 5-h-old Drosophila melanogaster embryos induces chromatin decondensation and nuclear formation from demembranated Xenopus sperm. Newly formed nuclei have a peripheral lamina, a double membrane, and structures resembling pore complexes. Indirect immunofluorescence analyses demonstrate the association of Drosophila lamins and DNA topoisomerase II with newly assembled nuclei.     

Visualization of intracellular trafficking of exogenous DNA delivered by cationic liposomes

To visualize the intracellular trafficking of exogenous DNAs delivered by cationic liposomes, rhodamine-labeled DNAs were transfected into NIH3T3 cells and observed by confocal laser microscopy. After 0.5- to 1-h incubations, the DNAs reached the nucleus with a much higher frequency than that expected from the cell division rate. This result suggests that DNAs can enter the nucleus in the presence of the nuclear membrane. Interestingly, some DNAs appeared to extend through the nuclear membrane in the aggregated form which were much larger than the nuclear pore complex. The DNAs which have passed through the nuclear membrane were stained with SYTO 24, a DNA labeling reagent. The stained part may be "naked" DNA that is free of lipids or proteins. This observation indicates that a complex containing DNA fuses with the nuclear membrane and then naked DNA is released into the nucleus.     

利用非洲爪蟾精子染色质和卵提取物在体外重建细胞核

应用非洲爪蟾去膜精子染色质和卵提取物成功地进行了细胞核本外重建。当精子染色质加入卵提取物后,首先发生染色质去浓缩作用,染色质整体结构膨胀;膜泡在膨胀的染色质外周聚集并逐渐彼此融合成双层膜;核孔复合体以某种未知方式组装入双层膜而形成核膜结构,并逐渐完全覆盖膨大的染色质,最终形成典型的间期核结构。     

Fate of DNA injected into early Drosophila embryos

Plasmid DNA injected into early Drosophila embryos becomes enclosed in nuclei or nucleus-like structures where it remains at least throughout embryonic development. The fraction remaining in the cytoplasm is gradually degraded. The nuclear fraction is converted to a high-molecular-weight form consisting largely but not exclusively of tandem oligomers. Some of it, however, can occasionally become integrated in the genome. Extensive DNA replication takes place but few, if any, molecules are able to initiate a second round of replication.     

Persistence and expression of circular DNAs encoding Drosophila amylase, bacterial chloramphenicol acetyltransferase, and others in Xenopus laevis embryos

We injected circular forms of several different DNAs into fertilized eggs of Xenopus laevis, and studied the persistence and expression of the injected DNAs during early embryonic development. When we injected plasmids which contained Drosophila amylase genes, the copy number of the injected DNA increased only slightly during cleavage, started to decrease at the blastula stage, then became very small at the tadpole stage. In such embryos, Drosophila amylase activity was detected at and after the gastrula stage. When we injected other kinds of circular DNAs (pX1r101A, cDm2055, pII25.1, pBR322, and pSP-64-L14), their copy number did not increase throughout the early stages. When circular plasmids that contained bacterial chloramphenicol acetyltransferase (CAT) genes were injected, their copy number usually did not increase, but sometimes, for unknown reasons, it increased extensively throughout the blastula to gastrula stages. In both cases, CAT enzyme activity started to be expressed during the blastula to gastrula stages and disappeared at the 2 day-old tadpole stage. The level of CAT enzyme activity was roughly proportional to the amount of CAT mRNA formed, and also to the copy number of injected genes. From these results, we concluded that in Xenopus embryos, exogenously-injected circular DNAs are preserved for the most part as circular DNAs, and that the increase in their copy number within the embryos is not prerequisite for the expression of their genetic information.     

Formation of the postmitotic nuclear envelope from extended ER cisternae precedes nuclear pore assembly

During mitosis, the nuclear envelope merges with the endoplasmic reticulum (ER), and nuclear pore complexes are disassembled. In a current model for reassembly after mitosis, the nuclear envelope forms by a reshaping of ER tubules. For the assembly of pores, two major models have been proposed. In the insertion model, nuclear pore complexes are embedded in the nuclear envelope after their formation. In the prepore model, nucleoporins assemble on the chromatin as an intermediate nuclear pore complex before nuclear envelope formation. Using live-cell imaging and electron microscope tomography, we find that the mitotic assembly of the nuclear envelope primarily originates from ER cisternae. Moreover, the nuclear pore complexes assemble only on the already formed nuclear envelope. Indeed, all the chromatin-associated Nup107-160 complexes are in single units instead of assembled prepores. We therefore propose that the postmitotic nuclear envelope assembles directly from ER cisternae followed by membrane-dependent insertion of nuclear pore complexes.     

Comparative studies on the structural organization of membrane-depleted nuclei and metaphase chromosomes

Interphase membrane-depleted nuclei and metaphase chromosomes were prepared in parallel with a nonionic detergent lysis procedure at low ionic strength. By flow microfluorometry we showed for the first time that cell lysates contain all stages of the cell cycle in the same proportions as the starting cell population. Morphologically intact membrane-depleted nuclei and metaphase chromosomes were isolated as non-aggregated structures on sucrose gradients. When analysed in the electron microscope, membrane-depleted nuclei that had been treated with 2M NaCl appeared as residual structures containing the pore complex-lamina layer attached to a halo of DNA filaments. In contrast, no distinct high salt-resistant structure was found with metaphase chromosomes. They formed a highly fragile network which disintegrated easily into small complexes connected with DNA filaments. High salt-resistant DNA-protein complexes were purified by Metrizamide density gradient centrifugation. The main difference in the protein composition of interphase and metaphase residual complexes was the presence in interphase of a protein triplet in the 60–75 kilodalton molecular weight range and its absence in metaphase. This protein triplet most likely corresponds to the lamins A, B, and C of the nuclear lamina. The combined results suggest that the main difference in the structural organization of interphase nuclei and metaphase chromosomes is the presence or absence of the pore complex-lamina layer.     

Chromosomes and their relationship to nuclear components during the cell cycle in Chinese hamster cells

Summary Chromosomes and their relationship to nuclear components during various phases of the cell cycle were studied with different fixation, embedding, and enzyme techniques. The results showed that interphase chromosomes may have oriented in such a way that a given locus became associated with the nuclear membrane. Some chromosomes also appeared to interact with the nucleolus. The nuclear matrix materials, however, were distributed between the chromosomes and formed a delineating boundary for the chromosomes. These matrix materials, furthermore, formed channel-like structures within the nucleus and towards the cytoplasm through their interaction with nuclear pore complexes. During mitosis, chromosomes were encapsulated with material that appeared to be derived from the matrix, disintegrated residues and fragments of the nuclear envelope, the lamina, and nucleolar material. These chromosome-associated materials seen in mitosis appeared to serve as foci for formation of new nuclear components in subsequent interphase.     

Changes in distribution of nuclear pores during differentiation of the male germ cells.

Changes in number of nuclear pores in different states of physiologica activity have been reported, but little is known about changing patterns of distribution in the course of cell differentiation. Pore distribution in male germ cells was studied in freeze fracture preparations of immature and mature rodent testis. As in other somatic cells, pores were uniformly and apparently randomly distributed in Sertoli cell nuclei. The nucleus of gonocytes and spermatogonia showed varying degrees of pore clustering. Spermatocytes invariably exhibited very striking pore aggregation with close hexagonal packing in pore-rich areas, and large pore-free areas. In early spermatids, pores appeared to be randomly distributed. As the acrosome formed and spread over the apical pole of the nucleus, pores disappeared ahead of its advancing margin and became more concentrated in the post-acrosomal region. The relationship of pore complexes to the chromosomes and the role of the fibrous lamina are discussed. The question as to whether the changing patterns observed involve movement of pores within fluid nuclear membranes, or a dissolution and reformation of new pores remains unanswered.     

Ultrastructure of free ribonucleoprotein complexes in spread mammalian nuclei

Mouse erythroleukemia cell nuclei obtained by three different methods were spread for electron microscopy under low ionic conditions. It was found that this procedure allows the observation of free large ribonucleoprotein (RNP) complexes released from the nuclei during the centrifugation. The morphology of these complexes was readily affected by the conditions of cell treatment and spreading. Two extreme forms of free nuclear RNP structures were obtained, both consisting of spherical particles with diameters of approximately 17-20 nm. The first type was of loosened complexes of irregularly assembled particles interconnected with RNA fibrils. The second represented tightly packed particles forming mostly branched structures. The latter structures appeared to be closer to the native form of the nuclear RNP particles, differing from polyribosomes by their characteristic branching and stability in EDTA solutions.     

Structure of the nuclear pore complex in mammalian cells. Two annular components

The ultrastructure of the nuclear pore complex has been investigated in isolated nuclei of an in vitro cultured bovine liver cell line. In shadow-cast replicas of the surface of nuclei isolated in Tris buffer containing low K⁺ and Mg²⁺ concentrations (RSB) the rims of the pores appeared as annular projections with an outer diameter of 100 to 120 nm. When the nuclei were isolated in Tris buffer containing 0.1% Triton the projections were essentially lost, together with the outer membrane of the nuclear envelope. In electron micrographs of whole-mount preparations the Triton-Tris nuclei—but not the RSB nuclei—were surrounded by numerous circular structures, which obviously had been detached from the nuclear surface during the preparation. They consisted of eight granules of about 20 nm diameter which were connected in a circular fashion by fibrous material. The circular structures had an inside diameter close to 65 nm. In broken nuclei many of these circular structures contained a second, smaller circular component and a central granule. From these observations it is concluded that the annulus of the nuclear pore consists of two components and that the outer component is located in the perinuclear space in intimate association with the membrane limiting the pore. A modified model of the nuclear pore complex which accounts for this location is proposed.