Nuclear Envelope Reformation Research Articles

Exogenous histone H1 injection into mitotic cells disrupts synchronous progression of mitotic events by delaying chromosome decondensation

ABSTRACT At the end of open mitosis, chromosome decondensation, nuclear envelope re-formation and reassembly of interphase microtubules following mitotic spindle dissociation occur coordinately. To determine whether these events progress only synchronously in vivo, we delayed chromosome decondensation by injecting of exogenous proteins into the mitotic rat kangaroo kidney epithelium (PtK2) cells. When histone H1 purified from calf thymus was injected at prometaphase, chromosome condensation was prolonged for several hours, and sister chromatid separation and cytokinesis did not occur. However, interphase microtubules reassembled and lamin B-positive structures re-formed around the condensed chromosomes. Exactly the same results were obtained on injection of bacterially expressed H1. Kinetic experiments showed that there were two types of lamin B-positive structures. One type (type A) was stained uniformly with anti-lamin B antibodies. The other (type B) showed peripheral lamin B staining; that is, the normal interphase staining pattern, and was found to be competent for nuclear protein transport. As the chromosomes decondensed, the amount of type A decreased and that of type B increased. However, even cells containing highly condensed chromosomes had both type A and type B. From these results, we conclude that the re-formation of microtubules and reassembly of a nuclear transport-competent envelope do not depend on chromosome decondensation.

Kinesin-like molecules involved in spindle formation

To study the possible involvement of kinesin-like molecules in mitosis a polyclonal antibody against the head domain of Drosophila kinesin heavy chain (HD antibody) was microinjected into PtK1 cells at the prophase-prometaphase transition. Progress of the cell through mitosis was recorded for subsequent detailed analysis. Cells injected with pre-immune IgG progressed through mitosis at rates similar to those for noninjected cells. After HD antibody injections, chromosomes failed to congress to an equatorial plane and cells failed to form a bipolar spindle. Rather, the spindle poles came together, resulting in a monopolar-like configuration with chromosomes arranged about the poles in a rosette. Sometimes the monopolar array moved to the margin of the cell in a way similar to anaphase B movement in normal cells. Antibody-injected cells progressed into the next cell cycle as evidenced by chromosome decondensation and nuclear envelope reformation. Anti-tubulin immunofluorescence confirmed the presence of a radial monopolar array of microtubules in injected cells. HD antibody stained in a punctate pattern in interphase and the spindle region in mitotic PtK1 cells. The antibody also reacted with spindle fibers of isolated mitotic CHO spindles and with kinetochores of isolated CHO chromosomes. Immunoblotting indicated that the major component recognized by the antibody is the 120 kDa kinesin heavy chain. At higher protein loads the antibody recognized also a 34 kDa polypeptide in PtK1 cell extracts, a 135 kDa polypeptide in a preparation of CHO spindles and a 300 kDa polypeptide in a preparation of CHO mitotic chromosomes. We conclude that a kinesin-like molecule is important for the formation and/or maintenance of the structure of mitotic spindle.

Ultrastructure of Mitosis and Cytokinesis In the Colorless Flagellate Katablepharis Ovalis Skuja

ABSTRACT. Mitosis and cytokinesis in Katablepharis ovalis, a colorless flagellate, was investigated. Two new flagella are produced prior to prophase, resulting in a motile quadriflagellate cell during mitosis. the inner array of microtubules of the feeding apparatus disappears before prophase begins. the nuclear envelope disperses during prophase, apparently being converted into rough endoplasmic reticulum. the chromatin condenses and the nucleolus disperses with spindle microtubules appearing oriented perpendicular to the longitudinal axis of the cell. At metaphase, the chromatin is condensed as a single disc‐shaped mass and rough endoplasmic reticulum flanks the chromatin mass on each side. Groups of spindle microtubules pass through tunnels in the rough endoplasmic reticulum and through electron‐translucent areas of the chromatin. the spindle microtubules end at a number of minipoles in the cytoplasm. Vesicles, ribosomes, mitochondria and endoplasmic reticulum migrate among the spindle microtubules. There is no polar body or any electrondense area associated with the spindle poles. the basal bodies of the flagella remain attached to the axonemes and do not participate in mitosis. In anaphase, the chromatin separates and migrates to the poles. During telophase, the nuclear envelope reforms from the rough endoplasmic reticulum and the nucleoli reappear. the spindle microtubules are persistent during telophase. Cytokinesis occurs by longitudinal fission, starting at the anterior end and progressing posteriorly. Cytokinesis may be driven by elongation of the spindle microtubules since there is no visible structure associated with the furrowing.

Effects of okadaic acid on mitotic HeLa cells.

Mitotic HeLa cells were treated with different concentrations of okadaic acid (OA), known to inhibit phosphatase 1 and 2A activities. The cytological effects on the course of mitosis were studied at the light microscopic, immunoflourescence and electron microscopic levels. At the lowest concentration used (1 nM), OA did not show any effect on mitosis, but at higher concentrations it showed pronounced effects. The mitotic chromosomes became scattered, the mitotic spindle became deranged and the cells failed to enter anaphase. At the electron microscopic level formation of isolated microtubules and regular trilaminar kinetochores were observed. An extensive growth of the endoplasmic reticulum could be noted in these cells. Decondensation of chromatin and nuclear envelope re-formation could be seen only after withdrawal of OA. A high frequency of multinucleate cells could be found after 24 h of recovery. Cells treated with 100 nM OA for 3 hours showed diplochromosomes in over 50% of mitotic cells after 24 h recovery. These were presumably formed due to the failure of sister chromatid separation in the earlier mitosis in the presence of OA. At the electron microscopic level the diplochromosomes showed a quadruplet structure. The role of phosphatase 1 in controlling some late mitotic events, i.e. sister chromatid separation, MPF-inactivation and nuclear envelope re-formation etc., is discussed.

A nucleolar auto-antigen is part of a major chromosomal surface component

Several nucleolar antigens are defined by human autoantibodies. These antigens can therefore be used to follow the fate of nucleolar components through mitosis when this major nuclear structure disintegrates and becomes reassembled in G1-phase. We found that fibrillarin leaves the nucleolus before complete breakdown of this structure and attaches to chromosomes before nuclear envelope breakdown. In mouse, fibrillarin attaches over the chromosomal surface except for the excluded centromeric region. The antigen is transported to the new nucleus via the chromosomes and is last seen on chromosomal surfaces facing the cytoplasm during nuclear envelope reformation. Lamin B reappears on the same chromosomal surfaces before the nucleolar antigen is removed and aggregates for new nucleolar reformation in G1-phase cells. From our observations, we postulate that the antigen acts in concert with other proteins as a nuclear envelope equivalent by forming a protective sheath around the chromosome, that it excludes larger molecules, and helps to separate the chromosomes, in addition to segregation of the ribonucleoprotein (RNP) back to the nucleus for nucleolar reconstruction. We also suggest that the selective retention of these antigens from certain areas on individual chromosomes together with specific lamin B attachment over these chromosomal surfaces allows for a nonrandom positioning of chromosomes in the nucleus.

On the cell-free association of lamins A and C with metaphase chromosomes

Nuclear envelopes have previously been shown to assemble spontaneously around endogenous chromosomes in cell-free homogenates of mitotic Chinese hamster ovary cells. In order to further analyze the mechanisms underlying nuclear envelope reformation and the functions of the individual nuclear lamin polypeptides, a fractionated cell-free nuclear envelope reassembly system involving purified chromosomes and either a postchromosomal supernatant or a cytosol fraction from mitotic cells has been devised. Results obtained with this fractionated system show that lamins A and C will associate with the surfaces of chromosomes in the absence of lamin B and membranes, this association being inhibitable by ATP-gamma-S. However, in the absence of membranes chromatin decondensation never occurs. Using the reversible swelling of chromosomes in low ionic strength buffers lacking divalent cations as the basis of a simple assay, it is demonstrated that the association of lamins A and C with the surfaces of chromosomes has a pronounced and easily observable effect on chromatin organization.

Functional role of newly formed pore complexes in postmitotic nuclear reorganization.

Many nuclear proteins are released into the cytoplasm at prometaphase and are transported back into the daughter nuclei at the end of mitosis. To determine the role of this reentry in nuclear remodelling during early interphase, we experimentally manipulated nuclear protein uptake in dividing cells. Recently we and others have shown that signal-dependent, pore complex-mediated uptake of nuclear protein is blocked in living cells on microinjection of the lectin wheat germ agglutinin (WGA), or of antibodies such as PI1 that are directed against WGA-binding pore complex glycoproteins. In the present study, we microinjected mitotic PtK2 cells with WGA or antibody PI1 and followed nuclear reorganization of the daughter cells by immunofluorescence and electron microscopy. The inhibitory effect on nuclear protein uptake was monitored by co-injection of the karyophilic protein nucleoplasmin. When injected by itself early in mitosis, nucleoplasmin became sequestered into the daughter nuclei as they entered telophase. In contrast, nucleoplasmin was excluded from the daughter nuclei in the presence of WGA or antibody PI1. Although PtK2 cells with blocked nuclear protein uptake completed cytokinesis, their nuclei showed a telophase-like completed cytokinesis, their nuclei showed a telophase-like organization characterized by highly condensed chromatin surrounded by a nuclear envelope containing a few pore complexes. These findings suggest that pore complexes become functional as early as telophase, in close coincidence with nuclear envelope reformation. They further indicate that the extensive structural rearrangement of the nucleus during the telophase-G1 transition is dependent on the influx of karyophilic proteins from the cytoplasm through the pore complexes, and is not due solely to chromosome-associated components.

Colchicine-resistant assembly of tubulin in plant mitosis

Tubulin distribution in c-mitoses (induced by 1 mM colchicine) has been studied by indirect immunofluorescence with monoclonal antibodies inAllium cepa L. meristems proliferating under steady state kinetics. Two hours after colchicine treatment was initiated tubulin is detected in approximately 25% of the cells as arrowheads on the kinetochores, as if these structures stabilize microtubules against disassembly. Total disassembly of microtubules occurs in 70% of the c-mitoses six hours after the initiation of the colchicine treatment, when restitution nuclei also start appearing. After 2 to 14 hours of colchicine treatment, tubulin is detected in about 30% of the c-mitoses, both in small kinetochores-like dots and in a strand which apparently connects sister kinetochores. Other larger microtubule-like structures, up to 20 μm long, apparently unassociated with kinetochores, are assembled in the presence of cholchicine in c-mitoses after 10 hours. Such structures disappear when chromosomes decondense and the nuclear envelope reforms in the restitution nucleus; they do not seem to be related to interphase cortical microtubules which reappear in control telophase.

Nuclear envelope reformation and chromosome decondensation are dissociable events

Cells treated with 2,4-dinitrophenol, a metabolic inhibitor, show a strong retardation of anaphase movement. At the ultrastructural level these cells reveal nuclear envelope reformation without concurrent decondensation of the chromosomes which indicates that these are possibly two dissociable late mitotic events.

Meiosis reinitiation in the mollusc Patella vulgata. Regulation of MPF, CSF and chromosome condensation activity by intracellular pH, protein synthesis and phosphorylation

Abstract The dependency of some key events of the cell cycle upon pH, phosphorylation and protein synthesis was investigated during meiosis reinitiation of Patella vulgata oocytes stimulated by ammonia. In this report, we show, through heterologous microinjection experiments, that a stable maturation promoting factor (MPF) is actually produced under these conditions. This factor, which may be amplified in recipient germinal vesicle (GV)-blocked oocytes of the starfish Asterias rubens, but not of Patella, remains present in the metaphase-1-blocked oocytes for at least 2·5 h. Using effective concentrations of the protein synthesis inhibitor emetine, we further demonstrate that the appearance of this factor does not depend on newly made proteins, whereas the maintenance of metaphase-1 conditions and chromosome condensation activity (CCA), which has been related to the simultaneous presence of a cytostatic factor (CSF), requires a continuous supply of new short-lived proteins. Without this, the chromosomes decondense and the nuclear envelope reforms. Finally, we show that the entry into M-phase is accompanied by extensive protein phosphorylation, while the drug 6-dimethylami-nopurine (6-DMAP), which induces protein déphosphorylation without affecting protein synthesis or phosphoprotein phosphatase activity, produces the same cytological effects as emetine. The fact that the effect of 6-DMAP, both on protein phosphorylation and chromosome structure, is perfectly reversible indicates that Patella CSF activity must also be controlled at the posttranslational level, possibly through the activation of relevant protein kinases.

Ultrastructure of Chilomonas paramecium and the phylogeny of the cryptoprotists

The ultrastructure of the cryptoprotist Chilomonas paramecium is reviewed and compared to earlier accounts. Distinctive features include a complex cytoskeleton which defines the cell organization and interconnects cell components; trichocysts which resemble those in other cryptoprotists; and two non-photosynthetic plastids. During mitosis there is partial dispersal of the nuclear envelope early in prophase but some remains at the nuclear periphery throughout mitosis. At metaphase chromosomes are arrranged on the longitudinal axis of an elgonated elliptical nucleus. During telophase the chromosomes decondense and the nuclear envelope reforms. Cell structure is compared with that in other cryptoprotists, and origin of this taxon of algae is discussed.

Is nuclear envelope re-formation an inducible event?

In large multinucleate cells the nuclei enter mitosis and reach metaphase almost synchronously by interaction of the different parts of the cell, but some degrees of postmetaphase asynchrony still persist. Apart from chromosome movements, the important postmetaphase events are re-formation of the nuclear envelope, chromosome decondensation, and back-formation of the spindle. From ultrastructural studies of multinucleate cells showing asynchronous mitotic progression beyond metaphase, we observed that nuclear envelope re-formation takes place nearly synchronously in all chromosome groups as soon as one group has reached telophase and while others are still in earlier mitotic stages. This indicates that nuclear envelope re-formation is an inducible event independent of the degree of condensation or decondensation of the chromatin and may depend on a factor(s) opposite in behavior to the maturation-promoting factor.

Pronamide disrupts mitosis in a unique manner

Pronamide (3,5-dichloro( N-1,1-dimethyl-2-propynyl)benzamide) has traditionally been grouped with mitotic disrupters, such as colchicine or trifluralin, that inhibit polymerization of tubulin into microtubules. Many of the effects of pronamide ( c-metaphases, polymorphic nuclei, isodiametric cells, abnormal xylem development, swollen root tips) are similar to these well-studied mitotic disrupters. However, immunofluorescence microscopy studies using anti-tubulin sera revealed that, unlike other mitotic disrupters, pronamide-treated cells have greatly shortened microtubules that are located only at the kinetochore region. These structures detected by immunofluorescence studies were determined by electron microscopy to be microtubules. Because these short kinetochore microtubules did not allow for mitosis to proceed normally, the nuclear envelope reforms around the chromosomes leading to polymorphic nuclei. Frequently, other cytoplasmic organelles normally excluded in the zone occupied by the chromosomes and spindle during mitosis were retained within the reformed nuclei. These data indicate that, although the net effect of pronamide is similar to those of colchicine and trifluralin, pronamide acts by a different mechanism.

The Ultrastructure of Meiosis in Woronina pythii (Plasmodiophoromycetes)

During cleavage of cystogenous plasmodia, nuclei of Woronina pythii underwent one meiosis I-like division. The division was centric, closed with polar fenestrations, and characterized by the formation of synaptonemal complexes. Pachytene nuclei exhibited nuclear projections which were heretofore undocumented structural modifications of the nuclear membrane. Hypothetical functions of these structures are proposed. During diakinesis, fragmentation of the nuclear envelope occurred at the poles through the apparent growth of microtubules. Intranuclear membranous vesicles appeared to bud from the inner membrane of the original nuclear envelope during metaphase and anaphase, and coalesced on the surfaces of the separating chromatin masses, thereby forming new daughter nuclear envelopes within the intact original nuclear envelope. Similar descriptions of the spindle apparatus and events relating to nuclear envelope breakdown and reformation have been reported for the mitotic divisions of W. pythii. Nuclei of some mature cysts contained intranuclear cisternae which were continuous with the inner membrane of the nuclear envelope, and these cisternae frequently partitioned the nuclei into two or three separate compartments. The formation of intranuclear cisternae was presumed to result in the eventual fission of nuclei, which may represent functionally the second meiotic division.

Cyclic assembly-disassembly of cortical microtubules during maturation and early development of starfish oocytes

An extensive array of cortical microtubules in oocytes of the starfish Pisaster ochraceus undergoes multiple cycles of disappearance and reapparance during maturation and early development. These events were studied in isolated fragments of the oocyte cortex stained with antitubulin antibodies for indirect immunofluorescence. The meshwork of long microtubules is present in the cortex (a) of immature oocytes, i.e., before treatment with the maturation-inducing hormone 1-methyladenine, (b) for 10–20 min after treatment with 1-methyladenine, (c) after formation of the second polar body (in reduced numbers in unfertilized oocytes), and (d) in the intermitotic period between first and second cleavage divisions. The array of cortical microtubules is absent in oocytes (a) undergoing germinal vesicle breakdown, (b) during the two meiotic divisions (polar body divisions), and (c) during mitosis of the first and, perhaps, subsequent cleavage divisions. The cycle of assembly-disassembly of cortical microtubules is synchronized to the cycle of nuclear envelope breakdown and reformation and to the mitotic cycle; specifically, cortical microtubules are present when a nucleus is intact (germinal vesicle, female pronucleus, zygote nucleus, blastomere nucleus) and are absent whenever a meiotic or mitotic spindle is present. These findings are discussed in terms of microtubule organizing centers in eggs, possible triggers for microtubule assembly and disassembly, the eccentric location of the germinal vesicle, and the regulation of oocyte maturation and cell division.

Interphase chromosome order: A proposal

A model for the arrangement of chromosomes in interphase nuclei is proposed. The model assumes that interphase chromosomes have a Rabl orientation (a relic telophase arrangement). During interphase and prophase telomeres are attached to the nuclear envelope often in pairs. The association of telomeres, homologous or nonhomologous, is based on similarity of arm lengths and occurs at the time the nuclear envelope reforms. At this stage arm lengths will vary to some extent due to the amount of uncoiling etc. The sequence of chromosomes resulting from telomere-telomere pairing may vary among cells, but the number of arrangements will be restricted by arm length similarities. The ramifications of this model on melotic pairing, the constant attachment of chromosomes to some structure throughout the cell cycle, the distribution of genes within nuclei, and chromosome evolution are raised.

The ultrastructure of mitosis during sporangiogenesis inWoronina pythii (Plasmodiophoromycetes)

Mitotic divisions during sporangiogenous plasmodial cleavage inWoronina pythii were studied with transmission electron microscopy. We conclude that these nuclear divisions (e.g., transitional nuclear division, and sporangial mitoses) share basic similarities with the cruciform nuclear divisions inW. pythii and other plasmo-diophoraceous taxa. The major distinction appeared to be the absence of nucleoli during sporangial mitosis and the presence of nucleoli during cruciform nuclear division. The similarities were especially evident with regard to nuclear envelope breakdown and reformation. The mitotic divisions during formation of sporangia were centric, and closed with polar fenestrae, and characterized by the formation of intranuclear membranous vesicles. During metaphase, anaphase, and telophase, these vesicles appeard to bleb from the inner membrane of the original nuclear envelope and appeared to coalesce on the surface of the separating chromatin masses. By late telophase, the formation of new daughter nuclear envelopes was complete, and original nuclear envelope was fragmented. New observation pertinent to the mechanisms of mitosis in thePlasmodiophoromycetes include a evidence for the incorporation of membrane fragments of the original nuclear envelope into new daughter nuclear envelopes, and b the change in orientation of paired centrioles during sporangial mitosis.

Evidence for a unique profile of phosphatidylcholine synthesis in late mitotic cells.

Evidence is presented that the structural rearrangements in late mitosis are accompanied by an alteration in membrane lipid synthesis. This evidence was derived from analyzing phospholipid classes after rapid-labeling, as well as from determining the intracellular site of incorporation of choline by HeLa S3 cells as they progressed from metaphase into early interphase (G1). Compared with postmitotic cell data, the recent mitotic cell data indicate a specific two- to threefold increase in the net synthesis of phosphatidylcholine (PC) species, which appeared to contain the more saturated fatty acids. Since this was observed with glycerol, choline, and orthophosphate labelings, and not with methyl labeling, it appears that the CDP-choline plus diacylglycerol pathway rather than the phosphatidylethanolamine to PC pathway was augmented. Electron microscope autoradiography of anaphase, telophase, and early G1 cells demonstrated that the reformed nuclear envelope was the incorporation site of a significant proportion of the newly synthesized PC. This incorporation occurred by early telophase prior to chromosome decondensation. The potential significance of PC metabolism with regard to membrane rearrangements, such as nuclear envelope reformation, is discussed.

Reformation of the nuclear envelope in melanophores during recovery from a hormone plus puromycin treatment

Previous experiments have shown that treatment of the melanophores of Pachymedusa (Agalychnis) dacnicolor with Melanophore-Stimulating Hormone (MSH) + puromycin causes the nuclear envelope to breakdown leading to the formation of discontinuous vesicles and the hyperdispersion of chromatin. We show here that these cells recover, reform their nuclear envelopes, and recondense their chromatin, both in the presence and in the absence of actinomycin D (actD). After recovery, these cells respond to MSH by melanosome dispersion. From these results, the following conclusions or observations are drawn: 1. 1, Reformation of nuclear envelope does not require that the chromatin be condensed into chromosomes as in mitosis. 2. 2, The new nuclear envelope is derived primarily from reutilization of the membrane vesicles produced during nuclear envelope breakdown, somewhat similar to mitosis. There may also be contributions from other membranous organelles. 3. 3, The hyperdispersed chromatin appears not to be subject to extensive attack by endogenous nucleases as the recovered cells are of good ultrastructure and can respond tropically to MSH. 4. 4, The presence of actD appears not to prevent the conversion of the hyperdispersed chromatin into the normal pattern.

On the ‘clock’ mechanism determining the time of tissue-specific enzyme development during ascidian embryogenesis II. Evidence for association of the clock with the cycle of DNA replication

ABSTRACT Acetylcholinesterase (AChE) is a tissue-specific enzyme of the muscle cells of ascidian embryos and its synthesis begins at the neurula stage. Embryos which had been permanently cleavage-arrested with cytochalasin B could develop AChE activity. The time of first AChE occurrence in embryos which had been arrested in the 32-cell stage with cytochalasin was about the same as in normal embryos. The nucleus in the cell of cytochalasin-arrested embryos divided in good synchrony with that of normal embryos. Embryos which had been continuously arrested with colchicine could also produce AChE activity at nearly the same time as did normal embryos. In the cell of colchicine-arrested embryos normal nuclear divisions did not occur, but the cell showed repeated cycles of nuclear envelope breakdown and nuclear envelope reformation in almost parallel with cell cycles of normal embryos. The cell of colchicine-arrested embryos incorporated [3H]thymidine. Aphidicolin, a specific inhibitor of DNA synthesis, prevented cleavages of ascidian eggs. Embryos which had been permanently arrested with aphidicolin in the cleavage stages up to the 64-cell stage did not develop AChE activity, while embryos which had been treated with it from the 76-cell stage onwards were found to be able to differentiate AChE activity. Based on these findings it was proposed that DNA replication is prerequisite for development of the histospecific protein and that the cycle of DNA replication is closely associated with the clock mechanism which is determining the time of initiation of the enzyme development.