PROPHASING OF INTERPHASE NUCLEI AND INDUCTION OF NUCLEAR ENVELOPES AROUND METAPHASE CHROMOSOMES IN HELA AND CHINESE HAMSTER HOMO- AND HETEROKARYONS

Fusing human HeLa metaphase cells with HeLa interphase cells resulted within 30 min in either of two phenomena in the resultant binucleate cell: either prophasing of the interphase nucleus or formation of a normal-appearing nuclear envelope around the metaphase chromosomes. The frequency of either occurrence was strongly dependent on environmental pH. At pH's of 6.6–8.0, prophasing predominated; at pH 8.5 nuclear envelope formation predominated. Additionally, the frequencies of the two events in multinucleate cells depended on the metaphase/interphase ratio. When the ratio was 0.33 nuclear envelope formation predominated; when it was 2.0 prophasing predominated. In their general features, the results with fused HeLa cells resembled those reported earlier with fused Chinese hamster Don cells. However, the results provided an indication that between pH 6.6 and 8.0 the HeLa metaphase cells possessed a much greater capacity than the Don metaphase cells to induce prophasing. Fusion of Don metaphase cells with HeLa interphase cells or of Don interphase cells with HeLa metaphase cells at pH 8.0 resulted in nuclear envelope formation or prophasing in each kind of heterokaryon. As in the homokaryons, the frequencies of the two events in the heterokaryons depended on the metaphase/interphase ratio. The statistics of prophasing and nuclear envelope formation in the homo- and heterokaryon populations were consistent with the notion that disruption or formation of the nuclear envelope depends on the balance attained between disruptive and formative processes.     

The Drosophila maternal-effect gene fs(1)Ya encodes a cell cycle-dependent nuclear envelope component required for embryonic mitosis

The maternal-effect gene fs(1)Ya is specifically required for embryonic mitosis in Drosophila. fs(1)Ya is involved in the initiation of the first embryonic mitosis and may also be necessary for subsequent embryonic mitotic divisions. fs(1)Ya encodes a 91.3 kd hydrophilic protein containing two putative MPF phosphorylation target sites and two potential nuclear localization signals. This protein is synthesized during postoogenic maturation from its maternal RNA and persists throughout embryogenesis. In early embryos, the fs(1)Ya protein is localized to the nuclear envelope from interphase to metaphase. During anaphase and telophase, it is dispersed in the nucleoplasm and cytoplasm, a behavior that is different from that of both the nuclear envelope and lamins. These results suggest that the fs(1)Ya protein is a cell cycle-dependent component of the nuclear envelope that specifically functions in embryonic mitosis.     

New patterns of nuclear lamina induced by cell fusion

During the eukaryote cell cycle the nuclear envelope displays a series of major morphogenetic changes, the most significant of which include its breakdown and reconstitution as cells move up to, pass through and emerge from division. The three polypeptides, lamins A, B and C, are major components of the nuclear pore complex-lamina fraction of the nuclear envelope and their association with the nuclear membrane or their dispersal in the cytoplasm reflects the existing balance between polymerization and depolymerization in the envelope. We have perturbed the lamina polymerization cycle by means of cell fusion between mitotic and interphase cells, following the redistribution of nuclear lamina protein by means of immunofluorescence techniques. In these heterophasic heterokaryons changes in the distribution of lamina occur as a function of (1) the time elapsed after fusion; (2) the ratio of mitotic to interphase elements in the cell, and (3) the stage in the cell cycle occupied by the interphase partner at the time of fusion. Depolymerization of nuclear lamina occurs most rapidly in cells with high ratios of mitotic to interphase elements, and especially in G1 rather than S-phase nuclei. While lamina depolymerization predominates early after fusion, at later times lamina is deposited around both the original metaphase and interphase nuclear masses and this is associated with the resumption of interphase activity in the form of limited DNA synthesis. These observations lead us to conclude that lamina depolymerization is under positive control mediated by diffusible factors in the cytoplasm of the metaphase partner. Repolymerization is likely to be associated with the inactivation of these factors as the heterokaryons age and, as a result, pass into an interphase-like state.     

INDUCTION OF NUCLEAR ENVELOPES AROUND METAPHASE CHROMOSOMES AFTER FUSION WITH INTERPHASE CELLS

The process of cellular fusion induced by Sendai virus in Chinese hamster cells (Don line) afforded us the opportunity to study nuclear envelope formation around metaphase sets in the presence of interphase nuclei, when chromosome pulverization failed to occur in such multinucleate cells. Morphologically, the enveloped metaphase chromosomes resembled a normal telophase nucleus, though minor differences prompted us to call it telophase-like. Electron microscopic observations demonstrated that the membranes enveloping the chromosomes appeared to be identical with a normal nuclear envelope. The longer the cells were incubated with Colcemid before fusion, the higher was the number of cells with telophase-like nuclei and the lower the percentage of cells with pulverizations. Furthermore, the number of pulverizations bore a somewhat direct relationship to the ratio of metaphase to interphase nuclei in multinucleate cells, and the number of telophase-like nuclei was inversely proportional to this ratio. A hypothesis is advanced in which a balance between the activities of a chromosome pulverization factor and a nuclear envelope formation factor, the former in metaphase cells and the latter in interphase cells, is decisive as to the nature of morphologic events observed in virus-induced fused cells.     

NuMA: a protein involved in nuclear structure, spindle assembly, and nuclear re-formation

The abundant coiled-coil protein NuMA is located in the nucleus during interphase, but when the nuclear envelope disassembles in prometaphase it rapidly redistributes to the developing spindle poles. Microinjection of antibodies to NuMA at or before metaphase can block spindle assembly or cause spindle collapse, indicating a role for NuMA in spindle function. NuMA must also play a key role in telophase, as NuMA antibodies or truncations of NuMA cause aberrant nuclear reassembly despite apparently normal chromosome segregation. Consistent with a structural role for NuMA in the nucleus, immunoelectron microscopy reveals NuMA to be a component of nuclear filaments.     

The GTPase Ran regulates chromosome positioning and nuclear envelope assembly in vivo

The GTPase Ran is known to regulate transport of proteins across the nuclear envelope. Recently, Ran has been shown to promote microtubule polymerization and spindle assembly around chromatin in Xenopus mitotic extracts and to stimulate nuclear envelope assembly in Xenopus or HeLa cell extracts. However, these in vitro findings have not been tested in living cells and do not necessarily describe the generalized model of Ran functions. Here we present several lines of evidence that Ran is indispensable for correct chromosome positioning and nuclear envelope assembly in C. elegans. Embryos deprived of Ran by RNAi showed metaphase chromosome misalignment and aberrant chromosome segregation, while astral microtubules seemed unaffected. Depletion of RCC1 or RanGAP by RNAi resulted in essentially the same defects. The immunofluorescent staining showed that Ran localizes to kinetochore regions of metaphase and anaphase chromosomes, suggesting the role of Ran in linking chromosomes to kinetochore microtubules. Ran was shown to localize to the nuclear envelope at telophase and during interphase in early embryos, and the depletion of Ran resulted in failure of nuclear envelope assembly. Thus, Ran is crucially involved in chromosome positioning and nuclear envelope assembly in C. elegans.     

1,2-Dioctanoylglycerol accelerates or retards mitotic progression in Tradescantia stamen hair cells as a function of the time of its addition

We have treated living, intact stamen hair cells from the spiderwort plant, Tradescantia virginiana, with 0.5 microgram/ml or 60 micrograms/ml 1,2-dioctanoylglycerol, a potent and permeant activator of protein kinase C, and have observed the rates of progression of mitosis from prophase through anaphase. We have found that in addition to the concentration used, the time of initial treatment with 1,2-dioctanoylglycerol defines the response by the cells. The cells rapidly undergo nuclear envelope breakdown when this diglyceride is added in very late prophase, 0 to approximately 8 min prior to the time of normal nuclear envelope breakdown. Anaphase onset occurs 28 min after nuclear envelope breakdown, rather than after the 33 min interval observed in untreated cells. Rapid progression through metaphase is also observed if cells are treated with 0.5 microgram/ml 1,2-dioctanoylglycerol during prometaphase, up to 15 min after nuclear envelope breakdown. The addition of 0.5 microgram/ml 1,2-dioctanoylglycerol in late metaphase, approximately 26 min after nuclear envelope breakdown, results in sister chromatid separation slightly ahead of its normal time, 33 min after nuclear envelope breakdown, and in precocious cell plate vesicle aggregation, 3-5 min earlier than that observed in untreated cells. Treatment of cells with 60 micrograms/ml of 1,2-dioctanoylglycerol at any point during the interval from 0 to approximately 5 min prior to nuclear envelope breakdown results in precocious entry into anaphase. If cells are treated with either 0.5 microgram/ml or 60 micrograms/ml 1,2-dioctanoylglycerol earlier than 20 min before nuclear envelope breakdown, they do not enter mitosis, but instead revert to interphase without dividing. When 1,2-dioctanoylglycerol is added at other times during mitosis, the rate of subsequent mitotic progression is dramatically slowed; the cells require greater than 55 min to progress from nuclear envelope breakdown to anaphase onset, though once in anaphase, the cells progress onward to cytokinesis at normal rates. Treatments o of cells with 1,3-dioctanoylglycerol at any point during prophase, prometaphase, or metaphase are without effect on the rate of subsequent mitotic progression. The shifts in response by cells treated at specific times with 1,2-dioctanoylglycerol during mid- and late metaphase may be indicative of the existence of one or more regulatory switch points (i.e., checkpoints) just prior to anaphase onset.     

NUCLEAR ENVELOPE-ASSOCIATED RESUMPTION OF RNA SYNTHESIS IN LATE MITOSIS OF HELA CELLS

The restitution of RNA synthesis in cultures progressing from metaphase into interphase (G₁) has been investigated in synchronized HeLa S₃ cells by using inhibitors of macro-molecular synthesis and the technique of electron microscope autoradiography. The rate of incorporation of radioactive uridine into RNA approached interphase levels in the absence of renewed protein synthesis. In contrast, maintenance of this rate in G₁ was dependent upon renewed protein synthesis. Restoration of synthesis of heterogeneous nuclear RNA occurred under conditions that inhibited production of ribosomal precursor RNA. In autoradiographs of individual cells exposed to radioactive uridine, silver grains were first detected after nuclear envelope reformation at the periphery of the chromosome mass but before chromosomal decondensation. These data are consistent with the following interpretation. Multiple RNA polymerase activities persist through mitosis and are involved in the initiation of RNA synthesis in early telophase at sites on the nuclear envelope.     

Calcium- and cell cycle-dependent association of annexin 11 with the nuclear envelope

Annexin 11 is a widely expressed calcium- and phospholipid-binding protein that resides in the nucleoplasm in many cultured cell lines. This is in contrast to its most extensively characterized in vitro ligand, the small calcium-binding protein S100A6 (calcyclin), which is concentrated in the nuclear envelope. Here we have examined the significance of the association of annexin 11 and S100A6 by asking whether circumstances exist in which the two proteins occupy the same subcellular localization. First, we show that in both A431 and vascular smooth muscle cells, elevation of intracellular Ca2+ leads to translocation of annexin 11 from the nucleus to the nuclear envelope where it co-localizes with S100A6. We also demonstrate, using fusions of annexin 11 with green fluorescent protein, that whereas the C-terminal core domain of annexin 11 is essential for Ca2+ sensitivity, the N-terminal domain is required for targeting to the nuclear envelope. Second, we show that annexin 11 relocalizes to the nuclear envelope as A431 cells transit from early to mid-prophase. In late prophase, at the time of nuclear envelope breakdown, annexin 11 and S100A6 become intensely localized with lamina-associated polypeptide 2 to folds in the nuclear envelope. From metaphase to telophase S100A6 is degraded, but in late telophase annexin 11 associates with the reforming nuclear envelope before resuming a nucleoplasmic location in interphase. These results show that co-localization of annexin 11 and S100A6 at the nuclear envelope may be regulated either by elevation of intracellular Ca2+ or by cell cycle progression and provide the first evidence that these proteins may associate in vivo.     

Dynamic distribution of TTK in HeLa cells： insights from an ultrastructural study

Entry into mitosis is driven by signaling cascades of mitotic kinases. Our recent studies show that TTK, a kinetochore-associated protein kinase, interacts with CENP-E, a mitotic kinesin located to corona fiber of kinetochore. Using immunoelectron microscopy, here we show that TTK is present at the nuclear pore adjacent complex of interphase HeLa cells. Upon nuclear envelope fragmentation, TTK targets to the outermost region of the developing kinetochores of monoorient chromosome as well as to spindle poles. After stable attachment, throughout chromosome congression, TTK is a constituent of the corona fibers, extending up to 90 nm away from the kinetochore outer plate. Upon metaphase alignment, TTK departs from the kinetochore and migrates toward the centrosomes. Taken together, this evidence strongly supports a model in which TTK functions in spindle checkpoint signaling cascades at both kinetochore and centrosome.     

ELECTRON MICROSCOPY OF MITOSIS IN A RADIOSENSITIVE GIANT AMOEBA

Various aspects of the ultrastructure of the dividing nuclei in the large radiosensitive amoeba Pelomyxa illinoisensis are demonstrated. Evidence of nuclear envelope breakdown is presented, and membrane fragments are traced throughout metaphase to envelope reconstruction in anaphase and telophase. Annuli in the nuclear envelope and its fragments are shown throughout mitosis. During metaphase and anaphase some 15 to 20 mitochondria are aligned at each end of the spindle, and are called polar mitochondria. The radioresistant amoebae Pelomyxa carolinensis and Amoeba proteus do not have polar mitochondria, and Pelomyxa illinoisensis is unique in this regard. The shape of the P. illinoisensis interphase nucleoli differs from that in the two radioresistant species, and certain aspects of nucleolar dissolution in the prophase vary. Helical coils in the interphase nucleoplasm are similar to those in the radioresistant amoebae. A "blister" phase in the flatly shaped telophase nuclei of P. illinoisensis is described which is interpreted to be the result of a rapid nuclear expansion leading to the formation of the normal spherical interphase nuclei.     

Chromosome ends and the nuclear envelope at premeiotic interphase in the male grasshopper Brachystola magna by 3-D,E.M. reconstruction

During premeiotic interphase in the male grasshopper Brachystola magna the nucleus is divided into two nuclear envelope bound compartments, one containing the X chromosome and one the autosomes. — The autosomal compartment is characterized by an invaginated nuclear envelope with nuclear pores distributed throughout the envelope. In a polarized region of the cell the pericentric heterochromatic chromocenters are associated with the inner membrane of the envelope invaginations. In this species the chromosomes are telocentric (acrocentric?) and the pericentric heterochromatin marks the proximal chromosome ends. It is concluded that the chromosome ends are attached to the nuclear envelope at premeiotic interphase. — Comparisons are made between the present observations on chromosome arrangements and the nuclear envelope at premeiotic interphase to earlier observations at early meiotic prophase in the same species (Church, 1976). It is concluded that a rearrangement of both the proximal chromosome ends and the nuclear envelope occurs as cells enter meiotic prophase.     

Scaffold attachment of DNA loops in metaphase chromosomes

We have examined the higher-order loop organization of DNA in interphase nuclei and metaphase chromosomes from Drosophila Kc cells, and we detect no changes in the distribution of scaffold-attached regions (SARs) between these two phases of the cell cycle. The SARs, previously defined from experiments with interphase nuclei, not only are bound to the metaphase scaffold when endogenous DNA is probed but also rebind specifically to metaphase scaffolds when added exogenously as cloned, end-labeled fragments. Since metaphase scaffolds have a simpler protein pattern than interphase nuclear scaffolds, and both have a similar binding capacity, it appears that the population of proteins required for the specific scaffold-DNA interaction is limited to those found in metaphase scaffolds. Surprisingly, metaphase scaffolds isolated from Drosophila Kc cells contain both the lamin protein and a pore-complex protein, glycoprotein (gp) 188. To study whether lamin contributes to the SAR-scaffold interaction, we have carried out comparative binding studies with scaffolds from HeLa metaphase chromosomes, which are free of lamina, and from HeLa interphase nuclei. All Drosophila SAR fragments tested bind with excellent specificity to HeLa interphase scaffolds, whereas a subset of them bind to HeLa metaphase scaffolds. The maintenance of the scaffold-DNA interaction in metaphase indicates that lamin proteins are not involved in the attachment site for at least a subset of Drosophila SARs. This evolutionary and cell-cycle conservation of scaffold binding sites is consistent with a fundamental role for these fragments in the organization of the genome into looped domains.     

COMPARATIVE CYTOLOGY OF ULOTHRIX AND STIGEOCLONIUM1

This investigation describes the cytology of the ulotrichalean genera Ulothrix and Stigeoclonium. Cellular organization is similar to the degree that interphase cells of the 2 genera cannot be distinguished with certainly. In Stigeoclonium, the nuclear envelope becomes disrupted at the end of prophase, and centrioles enter the nucleoplasm. At metaphase the nuclear envelope is again intact, and some of the spindle tubules appear to be contiguous with the nuclear envelope. The spindle in Ulothrix is essentially open with, no attachment of spindle tubules to the nuclear envelope and with, centrioles on the spindle-cytoplasm interface at the spindle poles. Spindle poles are blunt in Stigeoclonium and pointed in Ulothrix. Cytokinesis is by cell plate formation in both genera, but there is no phragmoplast.     

ELECTRON MICROSCOPIC STUDIES OF MITOSIS IN AMEBAE : I. Amoeba proteus

Individual organisms of Amoeba proteus have been fixed in buffered osmium tetroxide in either 0.9 per cent NaCl or 0.01 per cent CaCl₂, sectioned, and studied in the electron microscope in interphase and in several stages of mitosis. The helices typical of interphase nuclei do not coexist with condensed chromatin and thus either represent a DNA configuration unique to interphase or are not DNA at all. The membranes of the complex nuclear envelope are present in all stages observed but are discontinuous in metaphase. The inner, thick, honeycomb layer of the nuclear envelope disappears during prophase, reappearing after telophase when nuclear reconstruction is in progress. Nucleoli decrease in size and number during prophase and re-form during telophase in association with the chromatin network. In the early reconstruction nucleus, the nucleolar material forms into thin, sheet-like configurations which are closely associated with small amounts of chromatin and are closely applied to the inner, partially formed layer of the nuclear envelope. It is proposed that nucleolar material is implicated in the formation of the inner layer of the envelope and that there is a configuration of nucleolar material peculiar to this time. The plasmalemma is partially denuded of its fringe-like material during division.     

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.     

Monoclonal antibody to a protein of the nucleus and mitotic spindle of mammalian cells

We report here the isolation of a monoclonal antibody, J17, that reacts with a conserved vertebrate protein antigen that is present in the spindle apparatus during mitosis but found within the nucleus during interphase. Immunofluorescence microscopy demonstrates that the J17 antigen is found in numerous punctate regions that are distinct from nucleoli. Furthermore, this antigen is not directly associated with kinetochores, the nuclear envelope, or with metaphase chromosomes. — Antibody J17 immunoprecipitates a single polypeptide of very high molecular weight (over 250000) from K562 human erythroleukemia cells pulse-labeled with ¹⁴C-leucine. This polypeptide is converted quantitatively to a stable 220-kilodalton product within one cellular generation. We discuss the possible relevance of this processing event for transport into the nucleus. The J17 antigen is synthesized throughout the cell cycle in Chinese hamster ovary cells.     

ELECTRON MICROSCOPIC STUDIES OF MITOSIS IN AMEBAE : II. The Giant Ameba Pelomyxa carolinensis

Dividing nuclei from the giant ameba Pelomyxa carolinensis were fixed in osmium tetroxide solutions buffered with veronal acetate to pH 8.0. If divalent cations (0.002 M calcium, magnesium, or strontium as chlorides) were added to the fixation solution, fibrils that are 14 mµ in diameter and have a dense cortex are observed in the spindle. If the divalent ions were omitted, oriented particles of smaller size are present and fibrils are not obvious. The stages of mitosis were observed and spindle components compared. Fibrils fixed in the presence of calcium ions are not so well defined in early metaphase as later, but otherwise have the same diameter in the late metaphase, anaphase, and early telophase. Fibrils are surrounded by clouds of fine material except in early telophase, when they are formed into tight bundles lying in the cytoplasm unattached to nuclei. Metaphase and anaphase fibrils fixed without calcium ions are less well defined and are not observably different from each other. The observations are consistent with the concept that spindle fibrils are composed of polymerized, oriented protein molecules that are in equilibrium with and bathed in non-oriented molecules of the same protein. Partially formed spindle fibrils and ribosome-like particles were observed in the mixoplasm when the nuclear envelope had only small discontinuities. Remnants of the envelope are visible throughout division and are probably incorporated into the new envelope in the telophase. Ribosome-like particles are numerous in the metaphase and anaphase spindle but are not seen in the telophase nucleus, once the envelope is reestablished, or in the interphase nucleus.