Nuclear Envelope Reformation Research Articles

MITOSIS IN THE OCTAFLAGELLATE PRASINOPHYTE,PYRAMIMONAS AMYLIFERA(CHLOROPHYTA)1

ABSTRACTCell division is described in the octaflagellate prasinophytePyramimonas amyliferaConrad and is compared in related genera. Basal bodies replicate at preprophase and move toward the poles. Cells remain motile throughout division. The nuclear envelope disperses and chromosomes begin to condense at prophase. Pairs of multilayered kinetochores are evident on the chromosomes of the metaphase plate. Spindle microtubules extending from the region of the basal bodies and rhizoplasts attach to the kinetochores or extend from pole to pole. Numerous vesicles and ribosomes have entered the nuclear region and the incipient cleavage furrow invaginates. The chromosomes move toward the poles at anaphase leaving a broad interzonal spindle between the two chromosomal plates. The nuclear envelope reforms first around the chromatin on the side adjacent to the spindle poles and later on the interzonal side. The cleavage furrow progresses into the interzonal spindle at telophase. By late telophase the nucleoli have reformed and the chromosomes have decondensed. The interzonal spindle has not been observed late in telophase. As the cleavage furrow nears completion the cells begin to twist and contort, ultimately separating the two cells.

Effects of divalent cations and glucose on mitotic-like events in fused interphase-metaphase cells

The effects of Ca 2+, Mg 2+ and glucose on the mitotic-like events of prophasing and telophasing were studied in Sendai virus-fused interphase-metaphase (I-M) Chinese hamster binucleate cells. At normal extracellular ion concentrations and neutral pH, about 80–90% of I-M binucleates show prophasing (nuclear envelope dissolution and chromatin condensation) of the I nucleus and 10–15% show telophasing (nuclear envelope reformation and chromatin decondensation) of the M nucleus. To study the effects of cellular divalent cations, cells, depleted of about 77 % of exchangeable cell Ca 2+ as determined by 45Ca 2+ studies, were incubated in different concentrations of Ca 2+ or Mg 2+ for 30 min prior to cell fusion. We found that relatively high concentrations of Ca 2+ or Mg 2+ (0.84 mM) were essential for prophasing and that in the presence of 10-fold less Ca 2+ or Mg 2+ (0.084 mM) the majority of binucleates showed telophasing. In contrast to a differential effect of divalent cations on the nuclear changes, we found that glucose metabolism was required for both prophasing and telophasing. Additionally, interruption of glucose metabolism in the M cell, but not in the I cell, prior to cell fusion depressed the prophasing frequency about 70%. Although we do not know how divalent cations and glucose function in prophasing and telophasing, we will discuss evidence which suggests that the effects are not mediated through secondary effects on membrane potential, by changes in intracellular concentrations of Na + or K +, by simple osmotic changes, or through inhibition of protein synthesis.

Role of spindle microtubules in the control of cell cycle timing.

Sea urchin eggs are used to investigate the involvement of spindle microtubules in the mechanisms that control the timing of cell cycle events. Eggs are treated for 4 min with Colcemid at prophase of the first mitosis. No microtubules are assembled for at least 3 h, and the eggs do not divide. These eggs show repeated cycles of nuclear envelope breakdown (NEB) and nuclear envelope reformation (NER). Mitosis (NEB to NER) is twice as long in Colcemid-treated eggs as in the untreated controls. Interphase (NER to NEB) is the same in both. Thus, each cycle is prolonged entirely in mitosis. The chromosomes of treated eggs condense and eventually split into separate chromatids which do not move apart. This "canaphase" splitting is substantially delayed relative to anaphase onset in the control eggs. Treated eggs are irradiated after NEB with 366-nm light to inactivate the Colcemid. This allows the eggs to assemble normal spindles and divide. Up to 14 min after NEB, delays in the start of microtubule assembly give equal delays in anaphase onset, cleavage, and the events of the following cell cycle. Regardless of the delay, anaphase follows irradiation by the normal prometaphase duration. The quantity of spindle microtubules also influences the timing of mitotic events. Short Colcemid treatments administered in prophase of second division cause eggs to assemble small spindles. One blastomere is irradiated after NEB to provide a control cell with a normal-sized spindle. Cells with diminished spindles always initiate anaphase later than their controls. Telophase events are correspondingly delayed. This work demonstrates that spindle microtubules are involved in the mechanisms that control the time when the cell will initiate anaphase, finish mitosis, and start the next cell cycle.

Chromatin influence on the function and formation of the nuclear envelope shown by laser-induced psoralen photoreaction.

Potorous tridactylis (PTK2) cells growing in culture were treated with psoralen derivatives and dividing cells were located by phase-contrast microscopy. Psoralens, light-sensitive DNA-photoadducting drugs, were reacted with mitotic chromosomes through exposure to 365-nm light from an argon laser microbeam system. It was found that following mitosis and photoreaction, cells without nuclear envelopes were produced when psoralen-treated cells received 60 light pulses over their entire chromosome complement. These 'non-nuclear membrane' cells were found to incorporate [3H]uridine and, to a lesser extent, [3H]thymidine by autoradiography. Reduction of the light exposure by half (30 near-u.v. pulses) over the entire chromosome complement in the presence of psoralen also produced non-nuclear-membrane cells as seen by light microscopy. Further examination of these cells (30 light pulses) by single-cell electron microscopy revealed that unlike the high light exposure (60 near-u.v. pulses), the low light dosage resulted in cells with membrane patches associated with their chromatin. Since neither actinomycin D nor cycloheximide impeded nuclear envelope reformation, the psoralen-DNA reaction is concluded to produce non-nuclear-membrane cells by a mechanism other than transcription or translation inhibition. The association of Golgi with areas of nuclear membrane patches gives indirect evidence of a possible Golgi contribution to the reformation of the nuclear envelope after mitosis. It is concluded that DNA plays a role in envelope reformation.

CRUCIFORM NUCLEAR DIVISION IN SOROSPHAERA VERONICAE

Sorosphaera veronicae Schroet. is an endobiotic, holocarpic, obligately parasitic fungus presently classified in the Plasmodiophoromycetes. The ultrastructure of nuclear envelope formation in somatic nuclear division in cystosoral plasmodia was studied. The inner membrane of the nuclear envelope during prophase appears to invaginate and blebb off intranuclear membranous vesicles. The intranuclear membranous vesicles become associated with the surface of the separating chromatin in anaphase and eventually are involved in the formation of daughter nuclear envelopes within the original nuclear envelope. The sequence of nuclear envelope breakdown and reformation in S. veronicae is noteworthy because it emphasizes alternate methods of nuclear envelope formation other than the generally considered “typical” formation described in Allium cepa L.

Cruciform Nuclear Division in Sorosphaera veronicae

Sorosphaera veronicae Schroet. is an endobiotic, holocarpic, obligately parasitic fungus presently classified in the Plasmodiophoromycetes. The ultrastructure of nuclear envelope formation in somatic nuclear division in cystosoral plasmodia was studied. The inner membrane of the nuclear envelope during prophase appears to invaginate and blebb off intranuclear membranous vesicles. The intranuclear membranous vesicles become associated with the surface of the separating chromatin in anaphase and eventually are involved in the formation of daughter nuclear envelopes within the original nuclear envelope. The sequence of nuclear envelope breakdown and reformation in S. veronicae is noteworthy because it emphasizes alternate methods of nuclear envelope formation other than the generally considered “typical” formation described in Allium cepa L.

Mitosis and cell division in Cryptomonas (Cryptophyceae)

Mitosis and cytokinesis are examined in Cryptomonas sp., a member of the Cryptophyceae. The beginning of prophase is signalled by the replication of the flagellar bases which are at the anterior of the cell and a proliferation of the microtubules which run from them to the nucleus at the posterior. The microtubules continue to proliferate as the flagellar bases migrate apart and the nucleus migrates to the anterior. They dissociate from the flagellar bases and enter the nucleus as the nuclear envelope breaks down. A rectangular spindle forms and at prometaphase chromatin is scattered along the spindle. From this stage until late telophase microtubules are found attached singly to the chromatin. In metaphase the chromatin forms a solid plate penetrated by tunnels through which microtubules pass and in anaphase the chromatin separates in two masses which move toward the poles as the spindle elongates. In telophase the nuclear envelope re-forms while a number of microtubules remain between the daughter nuclei. The cytokinetic furrow forms during metaphase and constricts gradually until cytokinesis is complete at telophase. A thin ringof amorphous material is seen under the plasma membrane in the cytokinetic furrow. These results suggest that on the basis of mitotic criteria there is little similarity between the cryptophytes and either the dinoflagellates or red algae to which they have been previously linked.

Ultrastructural changes in the nuclear envelope during mitosis of Chinese hamster cells: a proposed mechanism of nuclear envelope reformation.

Ultrastructural changes in the nuclear envelope during mitosis of Chinese hamster cells: a proposed mechanism of nuclear envelope reformation.

The effect of trifluralin on the ultrastructure of dividing cells of the root meristem of cotton (Gossypium hirsutum L. "Acala 4-42').

ABSTRACT Ultrastructural studies of trifluralin-treated cells in lateral root meristems of cotton (Gossy-pium hirsutum L.) revealed that mitotic disruptions were due to the absence of microtubules. The extent of disruption varied between individual roots and correlated with the presence or absence of microtubules. Where microtubules were absent, cells began division with a normal prophase chromosome cycle. The chromosomes did not line up along a metaphase plate, but coalesced in the cell. If cell division had begun prior to microtubule disappearance the mitotic process was arrested at the stage that had been reached when the disappearance occurred. In some cell divisions randomly oriented microtubules were noted, with mitosis apparently arrested at those stages. Nuclear envelope reformation yielded cells that were polyploid, polymorphonucleate, binucleate, or occasionally multinucleate. If microtubules were present and if their orientation were normal, all stages of mitosis occurred. The range of mitotic disruption observed can be explained by the threshold concentration for microtubule disappearance being very near aqueous saturation of trifluralin.

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 (G(1)) has been investigated in synchronized HeLa S(3) 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 G(1) 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.

Mitosis in heterobasidiomycetous yeasts. I. Leucosporidium scottii (Candida Scottii).

ABSTRACT Leucosporidium scottii, for many years considered to be an asporogenous yeast in the genus Candida, is now known to have a heterobasidiomycetous life-cycle. Although morphologically similar to ascomycetous yeasts such as Saccharomyces cerevisiae, budding cells of L. scottii have a strikingly different type of mitosis. In L. scottii, mitosis is not intranuclear as it is in 5. cerevisiae. Neither does the nucleus constrict and separate into daughter nuclei in the neck region between mother cell and bud. In L. scottii the chromatin-containing portion of the nucleus moves into the bud before division. There is partial breakdown of the nuclear envelope and the chromatin divides along a spindle apparatus formed inside the bud within remnants of nuclear envelope. A portion of the nucleus containing the nucleolus is left behind in the mother cell and disintegrates there when the envelope breaks down. After division, the nuclear envelope reforms around daughter nuclei and one daughter moves back to the mother cell. These events have been established on the basis of 3 types of observation: on living cells with phase-contrast microscopy; on cells stained for chromatin and studied with light microscopy; and on glutaraldehyde-fixed cells studied with electron microscopy. As in L. scottii, breakdown of nuclear envelope during division has been reported in several mycelial basidiomycetes. The sequence of chromatin movement into the bud of L. scottii, division inside the bud, and return of one daughter nucleus to the mother cell, is closely comparable to the movements of chromatin in and out of clamp connexions known to occur in dikaryotic hyphae of Polystictus versicolor. These mitotic similarities are in accord with the taxonomic relationship of L. scottii to the basidiomycetes. Observations on a cytoplasmic organelle located outside interphase nuclei of L. scottii have convinced us of its considerable importance during mitosis. This structure consists of 2 globular electron-dense ends joined by a bridge-like middle piece. It accompanies the advancing tips of nuclei which enter the bud. After breakdown of the nuclear envelope, the 2 spherical components of this organelle are seen at opposite poles of the mitotic spindle. Therefore we refer to it as the ‘microtubule organizing centre’ (MTOC). We have also speculated about a possible role for the organelle in the regulation of nuclear envelope growth, breakdown, and reformation.