The initiation of cell division in theChaetopterus Egg

Summary The various agents which can cause the initiation of cell division in the egg of the wormChaetopterus all cause an increase in the viscosity of the interior protoplasm. These agents include hypertonic solutions, isotonic potassium chloride solutions, cold, heat, acid, alkali, ether and ultraviolet radiation.This investigation was supported by a research grant from the National Cancer Institute, National Institutes of Health, Public Health Service.     

Mitotic waves and embryonic pattern formation: No correlation inCallosobruchus (Coleoptera)

Summary In early insect embryogenesis, mitosis (which is not accompanied by cell division) often starts at one or both egg poles and spreads like a wave over the egg. Relationships between these waves and those processes which coordinate spatial cell differentiation have been proposed. One possibility is that the egg region which has a slower mitotic rate may become temporally advanced in differentiation because of its longer interphase periods, so that the egg becomes polarized (Agrell 1962). Alternatively, the mitotic waves might reflect the position of different determined states (Kauffman 1973). We investigated the mitotic waves inCallosobruchus eggs, treated to produce 20% partially reversed segment sequences (double abdomens). In normal eggs, mitotic waves move predominantly from anterior to posterior whereas in treated eggs, the reversed posterior to anterior orientation was predominant. Despite this, we concluded that mitotic waves do not reflect processes involved in the specification of segment position because the reversal of mitotic waves was more than twice as frequent as the reversal of segment sequence and because they occurred in various control experiments in which there was no reversal of segment sequence.     

Isolation of acdc28 mutation that abrogates the dependence of S phase on completion of M phase of the budding yeast cell cycle

We have isolated a mutation in the budding yeastSaccharomyces cerevisisae CDC28 gene that allowscdc13 cells, carrying damaged DNA, to continue with the cell division cycle. Whilecdc13 mutant cells are arrested as largebudded cells at the nonpermissive temperature 37‡C, thecdc13 cdc28 double mutant culture showed cells with one or more buds, most of which showed apical growth. The additional buds emerged without the intervening steps of nuclear division and cell separation. We suggest that thecdc28 mutation abrogates a checkpoint function and allows cells with damaged or incompletely replicated DNA an entry to another round of cell cycle and bypasses the mitotic phase of the cell cycle.     

Temperature dependence of egg laying inAplysia brasiliana andA. californica

Summary The temperature dependence of egg laying was examined in winter-caughtAplysia. Cold-waterAplysia californica and warm-waterA. brasiliana were individually housed in the same large aquarium for 16 days at 15°C, and then for 16 days at 20°C. Initially, the majority of theA. californica were not reproductively mature (as determined by injections of atrial gland extracts) whereas all of theA. brasiliana were reproductively mature. When the temperature was increased from 15 to 20°C, both species showed a marked increase in the frequency of egg laying. At both temperatures,A. brasiliana laid eggs more frequently but produced smaller egg masses thanA. californica. We conclude that increased egg laying inA. californica was attibutable both to facilitation of oogenesis in previously reproductively immature animals and to increased activity of the bag cells which release an egg-laying hormone. Increased egg laying inA. brasiliana was attributable primarily to increased bag cell activity.     

Unequal first cleavage in the Tubifex egg: involvement of a monastral mitotic apparatus

The first cleavage in the freshwater oligochaete Tubifex hattai is unequal and meridional, and produces a smaller cell AB and a larger cell CD. This study traces the process of furrow formation, reorganization of cortical F-actin and the assembly of a mitotic apparatus during this unequal division. Cleavage furrow formation consists of two stages: (i) when eggs are viewed from the animal pole, meridionally running furrows emerge at two points of the egg's equator that are 90° apart from each other and approach the egg axis as they deepen; and (ii) at the midpoint between the equator and the egg center, the bottoms of these furrows link to each other on the animal and vegetal surfaces of the egg and form a continuous ring of constriction in a plane parallel to the egg axis. Egg cortices, isolated during the first step and stained with rhodamine-phalloidin, show that the bottoms of recently formed furrows are underlaid by a belt of tightly packed actin bundles (i.e. a contractile arc). The transition to the second stage of furrow formation coincides with the conversion of these actin belts into a continuous ring of F-actin. Whole-mount immunocytochemistry of microtubules reveals that the first cleavage in Tubifex involves an asymmetric mitotic spindle, which initially possesses an aster at one pole but not the other. This ‘monastral’ spindle is located at the egg's center and orients itself perpendicular to the egg axis. During anaphase, astral rays elongate to reach the cell surface, so that the array of astral microtubules in the plane of the egg's equator covers a sector of 270–300°. In contrast, it is not until the transition to telophase that microtubules emanating from the anastral spindle pole approach the cell margin. If eggs are compressed along the egg axis or forced to elongate, they form monastral spindles and divide unequally. In living compressed eggs, mitotic spindles, which are recognizable as bright streaks at the egg's center, appear not to shift their position along the spindle axis during division, suggesting that without eccentric migration of spindles Tubifex eggs are able to divide unequally. These results suggest that mechanisms that translocate the mitotic spindle eccentrically do not operate in Tubifex eggs during the first cell cycle. The mechanisms that generate asymmetry in spindle organization are discussed in the light of the present results.     

Über die Abgrenzung derChlorosarcinales von denChlorococcales

It is proposed to use amongst other characters the type of cell division in order to delimit theChlorosarcinales from theChlorococcales. A definition of the two processes of division occuring in these orders is given. It differs from that of other authors. In theChlorosarcinales only those genera should be assembled in which vegetative daughter cells arise by bipartition followed by firm association of the wall between the daughter cells with that of the mother cell. In contrast, autospores, the vegetative daughter cells of a number ofChlorococcales, develop by multiple division, their cell walls are formed all around the protoplasts and are free from that of the mother cell. The chlorococcalean generaTrebouxia andDictyochloropsis incorporate species which multiply by zoo-, aplano- and autospores as well as others having no autospores. Autospores possibly have arisen more than once during evolution.

     

Relationship between cell division and endogenous auxin in synchronously-cultured tobacco cells

In the cultured tobacco cell, we succeeded in obtaining a partial synchronization of cell division by a combination of pre-starvation, rhythmic light-dark pre-treatment and air tight pre-conditioning. The mitotic index increased during the light period according to the time interval after the end of pre-treatment, and reached its maximum (max=12%) at about 2.5 hr of irradiation, and about 80% of cells completed division 1.5 hr thereafter. During this period, IAA was biosynthesized in the cells, though these cells had been cultured in Murashige and Skoog's medium with 1 mg/l of 2,4-D as a growth substance. IAA was identified by paper chromatography, followed byAvena curvature test and combined gas chromatography-mass spectrometry. The time course of the increase and decrease in the amount of free IAA was parallel to that of the mitotic index. On the other hand, bound IAA increased later and decreased gradually after the end of cell division. Free IAA may have an important role in mitosis.     

Induction of paternal genome loss by the paternal-sex-ratio chromosome and cytoplasmic incompatibility bacteria (Wolbachia): A comparative study of early embryonic events

Paternal genome loss (PGL) during early embryogenesis is caused by two different genetic elements in the parasitoid wasp, Nasonia vitripennis. Paternal sex ratio (PSR) is a paternally inherited supernumerary chromosome that disrupts condensation of the paternal chromosomes by the first mitotic division of fertilized eggs. Bacteria belonging to the genus Wolbachia are present in Nasonia eggs and also disrupt paternal chromosome condensation in crosses between cytoplasmically incompatible strains. Cytoplasmic incompatibility Wolbachia are widespread in insects, whereas PSR is specific to this wasp. PGL results in production of male progeny in Nasonia due to haplodiploid sex determination. The cytological events associated with PGL induced by the PSR chromosome and by Wolbachia were compared by fluorescent light microscopy using the fluorochrome Hoescht 33258. Cytological examination of eggs fertilized with PSR-bearing sperm revealed that a dense paternal chromatin mass forms prior to the first metaphase. Quantification of chromatin by epifluorescence indicates that this mass does undergo replication along with the maternal chromatin prior to the first mitotic division but does not replicate during later mitotic cycles. Contrary to previous reports using other staining methods, the paternal chromatin mass remains condensed during interphase and persists over subsequent mitotic cycles, at least until formation of the syncytial blastoderm and cellularization, at which time it remains near the center of the egg with the yolk nuclei. Wolbachia-induced PGL shows several marked differences. Most notable is that the paternal chromatin mass is more diffuse and tends to be fragmented during the first mitotic division, with portions becoming associated with the daughter nuclei. Nuclei containing portions of the paternal chromatin mass appear to be delayed in subsequent mitotic divisions relative to nuclei free of paternal chromatin. Crosses combining incompatibility with PSR were cytologically similar to Wolbachia-induced PGL, although shearing of the paternal chromatin mass was reduced. Wolbachia may, therefore, block an earlier stage of paternal chromatin processing in the fertilized eggs than does PSR. © 1995 Wiley-Liss, Inc.     

Studying nuclear disassembly in vitro using Xenopus egg extract

Xenopus egg extract provides an extremely powerful approach in the study of cell cycle regulated aspects of nuclear form and function. Each egg contains enough membrane and protein components to support multiple rounds of cell division. Remarkably, incubation of egg extract with DNA in the presence of an energy regeneration system is sufficient to induce formation of a nuclear envelope around DNA. In addition, these in vitro nuclei contain functional nuclear pore complexes, which form de novo and are capable of supporting nucleocytoplasmic transport. Mitotic entry can be induced by the addition of recombinant cyclin to an interphase extract. This initiates signaling that leads to disassembly of the nuclei. Thus, this cell-free system can be used to decipher events involved in mitotic remodeling of the nuclear envelope such as changes in nuclear pore permeability, dispersal of membrane, and disassembly of the lamina. Both general mechanisms and individual players required for orchestrating these events can be identified via biochemical manipulation of the egg extract. Here, we describe a procedure for the assembly and disassembly of in vitro nuclei, including the production of Xenopus egg extract and sperm chromatin DNA.     

Asynchronous mitotic domains during blastoderm formation inMusca domestica L. (Diptera)

Summary We describe the mitotic cleavage patterns during blastoderm stage of the house flyMusca domestica L. Nuclear divisions up to mitotic stage 11 are apparently synchronous. Beginning with stage 12, nuclear divisions in the posterior third of the embryo lag behind, resulting first in a parasynchronous and finally in an asynchronous cleavage pattern. Thus a stage exists where all nuclei in the anterior region have completed 14 nuclear division cycles, while those in the posterior region have completed only 13 cycles. The border region between these nuclei is well defined and lies at 35% EL (egg length), the expression border of a gap gene. This border region is about 4–5 nuclei wide and shows a specialized mitotic behaviour.     

Mitotic cycle time and DNA content in annual and perennialMicroseridinae (Compositae,Cichoriaceae)

Within eight annual and perennialMicroseridinae species studied, the duration of the mitotic cycle is positively correlated with the nuclear DNA content, cycle time (hrs) = 7.3 + 0.32 × pg DNA/nucleus. Within the generaAgoseris andMicroseris, the annuals have lower DNA contents and more rapid mitotic cycle times than do the perennials. This relationship is predicted by the nucleotypic theory ofBennett. Annual species ofPyrrhopappus have relatively high DNA contents and a proportionately longer mitotic cycle time, but contrary to that expected by the nucleotypic theory as originally proposed have the fastest growth rate and shortest generation time observed in theMicroseridinae. This rapid developmental rate is discussed, nucleotypically, however, by analyzing relationships between DNA content, mitotic cycle time, and cell size.     

EFFECTS OF MICROTUBULE INHIBITORS ON PRONUCLEAR MIGRATION AND EMBRYOGENESIS IN FUCUS DISTICHUS(PHAEOPHYTA)1

Pronuclear migration in Fucus distichus spp. edentatus (de la Pyl.) Powell is blocked by incubation of fertilized eggs in colchicine (1 mg/ml) and Nocodazole (2 μg/ ml). Rhizoids form prior to decondensation of the sperm chromatin in eggs in which pronuclear fusion is blocked. This occurs during continuous colchicine incubation as well as in eggs recovering from a short treatment with either drug following fertilization. During recovery of the cells, the sperm and egg chromosomes condense, and the sperm chromosomes migrate toward the egg pronucleus. The delay in migration following removal of colchicine is as much as 24 h and is even slower following removal of Nocodazole. The egg chromosomes form a metaphase plate in treated cells while the sperm chromosomes are still distant in the cytoplasm. This suggests that egg centrioles are important in the mitotic division of the zygote, not sperm centrioles. The effect of colchicine treatment on the mitotic plane and cytokinesis is also discussed.     

Cyclin A, cyclin B and stringlike are regulated separately in cell cycle arrested trochoblasts of Patella vulgata embryos

 Trochoblasts are the first cells to differentiate during the development of spiralian embryos. Differentiation is accompanied by a cell division arrest. In embryos of the limpet Patella vulgata, the participation of cell cycle-regulating factors in trochoblast arrest was analysed as a first step to unravel its cause. We determined the cell cycle phase in which the trochoblasts are arrested by analysing the subcellular locations of mitotic cyclins. The results show that the trochoblasts are most likely arrested in the G2 phase. This was supported by measurement of the DNA content in trochoblast nuclei after the last division. Trochoblasts complete their final division at the sixth mitotic cycle. This mitotic cycle resembles the first postblastoderm cell cycle of Drosophila, in which mitotic activity is controlled by expression of the string gene. As failure of string expression results in cell cycle arrest in the G2 phase, negative regulation of a Patella string homolog could be responsible for trochoblast arrest. Although Stl messengers disappeared from trochoblasts during their final division, expression was observed again 20 min later. Messengers remained present in all trochoblasts at low levels during further development. Thus, expression of the stringlike gene allows the cell cycle arrest of these cells, whereas in Drosophila cells arrested in division lack string messengers. Received: 10 February 1997 / Accepted: 23 November 1997     

Light-induced synchrony of cell division in the protonema of the fern,Pteris vittata

Summary In protonemata of Pteris vittata grown for 6 days under red light, which brings about a marked depression of mitotic activity, the first division of the cells was synchronously induced by irradiation with blue light, and subsequent cell divisions were also promoted. The peak of the mitotic index reached a maximum of about 70% at 11.5 hrs, and 90% of all protonemata divided between the 11th and 13th hour after exposure to blue light. When the protonemata were continuously irradiated with blue light, synchronism of the next cell division in the apical cells decreased to a mitotic index of about 30%, and further divisions occurred randomly.The synchronization of cell division was found to be a combined effect of red and blue light. Red light maintained the cells in the early G₁ phase of the cell cycle; blue light caused the cells to progress synchronously through the cell cycle, with an average duration of 12 hr. By using ³H-thymidine, the average duration of the G₁, S, G₂ and M phases was determined to be about 3.5, 5, 2.5 and 1 hr, respectively.Synchronous cell division could be induced in older protonemata grown for 6 to 12 days in red light and even in protonemata having two cells. It could be repeated in the same protonema by reexposure to red light for 24 hrs or more before another irradiation with blue light.     

The effect of cobalt on the structure of chromosomes and on the mitosis

Summary Cobalt exerts an effect on the structure of chromosomes and on the course and intensity of cell division in root tips of Vicia faba is.Despiralisation of chromosomes and an increase of the viscosity of the matrix substance causing stickiness of the chromosomes are induced by the treatment with cobalt.A cytostatic effect of cobalt was observed. The intensity of cell division is strikingly reduced after treatment with cobalt. Cobalt inhibits the passage of interphase cells into prophase so that this element can be considered to be a preprophase poison.     

A twinfilin‐like protein coordinates karyokinesis by influencing mitotic spindle elongation and DNA replication in Leishmania

Twinfilin is an evolutionarily conserved actin‐binding protein, which regulates actin‐dynamics in eukaryotic cells. Homologs of this protein have been detected in the genome of various protozoan parasites causing diseases in human. However, very little is known about their core functions in these organisms. We show here that a twinfilin homolog in a human pathogen Leishmania, primarily localizes to the nucleolus and, to some extent, also in the basal body region. In the dividing cells, nucleolar twinfilin redistributes to the mitotic spindle and remains there partly associated with the spindle microtubules. We further show that approximately 50% depletion of this protein significantly retards the cell growth due to sluggish progression of S phase of the cell division cycle, owing to the delayed nuclear DNA synthesis. Interestingly, overexpression of this protein results in significantly increased length of the mitotic spindle in the dividing Leishmania cells, whereas, its depletion adversely affects spindle elongation and architecture. Our results indicate that twinfilin controls on one hand, the DNA synthesis and on the other, the mitotic spindle elongation, thus contributing to karyokinesis in Leishmania.     

THE FINE STRUCTURE OF OOGENESIS IN MARCHANTIA POLYMORPHA

Archegonium development, beginning with the archegonial initial and culminating in the mature egg, was studied with the electron microscope. The ultrastructural features of the beginning stages in development of the archegonium are relatively similar to one another. Plasmodesmata occur between all adjacent cells at this time. After the secondary central cell is formed these protoplasmic connections are lost, and both axial and parietal cell lineages begin to show signs of ultrastructural differentiation. The mature egg is characterized by cytoplasm rich in ribosomes and larger organelles. Mitochondria and simplified plastids commonly display a juxtaposed association. As far as could be ascertained the numerous plastids and mitochondria in the egg of Marchantia arise through division of preexisting organelles and are not formed anew from evaginations of the nucleus. Blebbing of the nucleus produces polymorphic organelles which appear to be pinched off into the cytoplasm. The mature egg also contains vacuoles and lipid bodies toward its periphery, while dictyosomes and extensive endoplasmic reticulum occur throughout. The space between the wall cells and the mature egg appears to contain an amorphous substance. No extra membrane was observed around the mature egg.     

Female reproductive development and pollen tube growth in diploid genotypes of<Emphasis Type="Italic"> Solanum cardiophyllum</Emphasis> Lindl.

We have characterized female gametophyte (megagametophyte) development and the kinetics of pollen tube growth in self-pollinated diploid genotypes (2n=2x=24) of Solanum cardiophyllum Lindl. that show normal seed formation. In this species megasporogenesis and megagametogenesis give rise to a female gametophyte of the Polygonum type composed of two synergids, an egg cell, a binucleated central cell and three antipodals; however, asynchronous abnormalities resembling mechanisms that prevail during the formation of second division restitution gametes were observed. In self-pollinated pistils at least 1–2% of germinating pollen tubes were able to reach the megagametophyte 60–84 hours after pollination (hap). Although the egg cell acquired a zygote-like morphology 60–84 hap, division of the primary endosperm nucleus was only observed 84 hap. The analysis of genetic variability in full-sib progeny confirmed that seeds are derived from sexual reproduction. These observations suggest that diploid genotypes of S. cardiophyllum can serve as an ideal system to genetically investigate true seed formation in a tuber-bearing Solanum species.