Ionic coupling and mitotic synchrony of siblings in a Drosophila cell line

Following mitosis in many cell lines, siblings remain adjoined in dyads until further cell division. We report here a series of experiments designed to ascertain the nature of this apposition in the embryonic Kc cell line of Drosophila melanogaster. We have found that (1) cell division in siblings is highly synchronized when compared to that in nonsiblings: (2) siblings in dyads are dye coupled with respect to Lucifer Yellow, but intercellular diffusion of larger molecules (FITC-dextran at 6 and 24 kDa) is retarded: (3) siblings are electrically coupled by an ungated low-resistance intercellular connection which is resistant to treatment with octanol and CO2, both known to close gap junction channels: and (4) members of a dyad are joined by a cytoplasmic bridge. Structures resembling septate junctions are also found between siblings and between cells in aggregates. The evidence accumulated here suggests that cytokinesis in Kc dyads is incomplete, resulting in an intercellular pathway that may provide for the passage of a molecular or electrical signal that regulates subsequent mitosis.     

Symmetrical segregation of potassium channels at cytokinesis

To determine how voltage-gated ion channels segregate between sibling cells at cytokinesis, we used a whole-cell patch clamp to measure the electrophysiological phenotypes of siblings within 45 min of division. Recently born siblings in an immortalized line of embryonic retinal cells were identified as pairs of spherical cells adhering to one another. All siblings were electrically coupled when cells were simultaneously voltage clamped, whereas nonsiblings were not coupled. Twelve paris of siblings were electrically isolated by mechanical separation so that their phenotypes could be measured independently. Cells expressed two principal membrane conductances, delayed rectifier-like (I_K) and inward rectifier (I_K(IR)) potassium currents. Despite qualitative and quantitative variability in I_K and I_K(IR) expression within the population, each cell of a given pair expressed similar steady-state current densities between –110 and +50 mV. We estimated I_K(IR) slope conductance by blocking the current specifically with 5 mM Cs and calculated I_K(IR) ratios in siblings and nonsiblings. Three pairs of siblings expressed I_K(IR) ratios of approximately 1.2, while ratios in three pairs of adhered nonsiblings varied between 1.6 and 5.4. When currents were sampled continuously through cytokinesis by using the perforated-patch recording mode, current amplitude showed no net change within 30 min of division. Because channel number did not appear to change in siblings during this interval, parental channels were inherited by each daughter in proportion to the area of membrane received. Heterogeneity therefore arises after siblings reenter interphase and is not due to the asymmetrical segregation of channels at cytokinesis. © 1993 John Wiley & Sons, Inc.     

The sphingosine 1-phosphate receptor 5 and sphingosine kinases 1 and 2 are localised in centrosomes: Possible role in regulating cell division

We show here that the endogenous sphingosine 1-phosphate 5 receptor (S1P₅, a G protein coupled receptor (GPCR) whose natural ligand is sphingosine 1-phosphate (S1P)) and sphingosine kinases 1 and 2 (SK1 and SK2), which catalyse formation of S1P, are co-localised in the centrosome of mammalian cells, where they may participate in regulating mitosis. The centrosome is a site for active GTP–GDP cycling involving the G-protein, G_i and tubulin, which are required for spindle pole organization and force generation during cell division. Therefore, the presence of S1P₅ (which normally functions as a plasma membrane guanine nucleotide exchange factor, GEF) and sphingosine kinases in the centrosome might suggest that S1P₅ may function as a ligand activated GEF in regulating G-protein-dependent spindle formation and mitosis. The addition of S1P to cells inhibits trafficking of S1P₅ to the centrosome, suggesting a dynamic shuttling endocytic mechanism controlled by ligand occupancy of cell surface receptor. We therefore propose that the centrosomal S1P₅ receptor might function as an intracellular target of S1P linked to regulation of mitosis.     

Mitosis in flat PTK2-human hybrid cells

The fusion of G0 human fibroblasts with PTK₂ (Potorous tridactylis) cells resulted in the production of hybrid heterokaryotic cells which remained flat in cell division. These cells permitted studies of mitosis in living hybrid cells without the need for fixation and staining. The breakdown of nuclear envelopes during prophase in a hybrid heterokaryotic cell correlated with the onset of premature chromosome condensation (PCC) in other nuclei in the same cell. Nuclear morphology and autoradiography demonstrated that the nuclei exhibiting PCC were from the human parent cells. Observation of multinucleated PTK₂-human hybrids in the later stages of mitosis showed that these cells normally produced three daughters instead of the usual two. Electron microscopic examination of dividing hybrid cells showed that the number of daughter cells was not related to the number of centrioles. Hybrid cells normally were found to contain many centriolar duplexes although not all of these structures were associated with active poles in mitosis. Cells with as many as six centriolar duplexes were found in mitosis. The configuration of the chromosomes in metaphase was found to be a more accurate indication of the number of daughters produced by a single division than the number of centrioles. Chromosome elimination in hybrid cells could also be visualized in PTK₂-human hybrids. Lagging chromosomes were commonly observed during mitosis and were often trapped in the constricting midbody.     

NUCLEAR BEHAVIOR IN THE VEGETATIVE HYPHAE OF CERATOCYSTIS FAGACEARUM

Vegetative nuclear division in Ceratocystis fagacearum (Bretz) Hunt was found to differ from classical mitosis in that: (1) division always occurs perpendicular to the longitudinal axis of the cell, (2) anaphase movement is unilateral and unsynchronized, (3) a spindle occurs only between separating chromatids. Interphase and prophase nuclei and nucleoli are morphologically similar to those in higher plants. At metaphase the associated chromosomes form a bar of chromatin and lie against the hyphal wall. Spindle fibers appear between separating chromatids, perhaps pushing them apart. When nuclear division is complete the nuclei become attenuated and migrate. Vegetative nuclear division in C. fagacearum may be an evolutionary form of classical mitosis.     

LIGHT-DEPENDENT INHIBITION OF CELL DIVISION AND RHIZOID ELONGATION IN GEMMAE OF THE FERN,VITTARIA GRAMINIFOLIA

Gametophytes of the shoe-string fern Vittaria graminifolia produce linear, six-celled propagules called gemmae. The terminal cells of each gemma elongate into primary rhizoids in culture, and the inner body cells divide asymmetrically to produce prothallial or rhizoid initials. The initiation of both asymmetric cell division and rhizoid elongation is delayed by light intensities greater than 2 w/m². The maximal rates of cell division and rhizoid elongation are unaltered. A 24-hr pulse of high light intensity delays cell division and rhizoid elongation to the same extent, whenever applied during the first 3 d of culture. The model we propose for cell division hypothesizes the existence of a preparatory phase of finite duration prior to mitosis that is sensitive to light intensity. If a cell is irradiated by light intensities greater than 2 w/m² while in the preparatory phase, its entrance into mitosis is delayed. A similar model is proposed for the initiation of rhizoid elongation. Despite the fact that both cell division and rhizoid elongation are dependent on photosynthesis, direct measurements of CO₂-uptake rates show that the inhibitory effects of high light intensities are not due to an inhibition of photosynthesis.     

Dynamics of spreading of cells of L-929 line after the mitosis

Using time-lapse microscopy, the changes in L-929 cells shape were analyzed during a cell cycle. During this time the cells were established to pass through three spreading stages. The highest rate of the cell spreading was observed during the first 1.5 h of mitosis. In this period, the cell area increases approximately 3-3.5 times following sigmoid dependence. After a short plateau the augmentation of the cell area starts also as a sigmoid dependence. This period is longer (up to 6 h after the beginning of cell division) with an additional 1.5-fold augmentation of the cells size. Next, the augmentation of the cells area goes linearly up to the beginning of the following mitosis. After the mother L-929 cell division, the daughter cells remained to be bridged together in the fission furrow site almost in 100% cases. The structure known as an intercellular bridge is related to a late telophase. In this connected state the L-cells are spreading and migrating up to 2.13 +/- 0.06 h where upon they are separated. Transition of the daughter cells from a round shape to the spread one occurring with the simultaneous maintenance of the intercellular bridge during a strictly determined time allows us to consider this phenomenon as independent and not relating to mitosis. We suggest naming this junction between the daughter cells as the "posttelophase intercellular bridge".     

Rab35 GTPase and OCRL phosphatase remodel lipids and F-actin for successful cytokinesis

Abscission is the least understood step of cytokinesis. It consists of the final cut of the intercellular bridge connecting the sister cells at the end of mitosis, and is thought to involve membrane trafficking as well as lipid and cytoskeleton remodelling. We previously identified the Rab35 GTPase as a regulator of a fast recycling endocytic pathway that is essential for post-furrowing cytokinesis stages. Here, we report that the phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) 5-phosphatase OCRL, which is mutated in Lowe syndrome patients, is an effector of the Rab35 GTPase in cytokinesis abscission. GTP-bound (active) Rab35 directly interacts with OCRL and controls its localization at the intercellular bridge. Depletion of Rab35 or OCRL inhibits cytokinesis abscission and is associated with local abnormal PtdIns(4,5)P2 and F-actin accumulation in the intercellular bridge. These division defects are also found in cell lines derived from Lowe patients and can be corrected by the addition of low doses of F-actin depolymerization drugs. Our data demonstrate that PtdIns(4,5)P2 hydrolysis is important for normal cytokinesis abscission to locally remodel the F-actin cytoskeleton in the intercellular bridge. They also reveal an unexpected role for the phosphatase OCRL in cell division and shed new light on the pleiotropic phenotypes associated with Lowe disease.     

A quantitative theory of liver regeneration in the rat. II: matching an improved mitotic inhibitor model to the data

A model of liver regeneration is put forward in which the rate of liver growth is controlled both by a liver-produced mitotic inhibitor and by the availability of parenchymal cells to enter the mitotic cycle. The model can be expressed as a pair of coupled differential equations, the first describing the dependance of inhibitor concentration on liver size and inhibitor decay and the second specifying the dependance of liver growth on inhibitor concentration and entry of cells into the mitotic cycle. The model is tested by comparing its solutions to the published data on mitotic indices following partial hepatectomy. For such a comparison, it is necessary to specify the cell-cycle time and the inhibitor dose-response function and half-life. If a negative exponential dose-response function, an inhibitor half-life of 11·4 h, and a cycle time of 18·25 h are postulated, the solutions match the data of Fabrikant (1968) who found that there were two waves of mitosis with a period of quiescence between them. The data of Grisham (1962), characterized by a single peak of mitosis, is matched by the theory using similar inhibitor properties but a shorter cell-cycle time (13·25 h); this causes the two peaks to overlap. In both cases, a better fit is obtained if the second cell cycle is longer than the first by 2–3 h. This suggests that cells enter a G₀ period after mitosis. A mechanism for littoral cell division, which occurs some 24 h after parenchymal cell division, is put forward in which the former cells depend on the enlargement of the latter for the stimulus to divide.     

Timing of micronuclear mitosis and its relation to commitment to division inParamecium tetraurelia

This study examines the timing of micronuclear mitosis during the vegetative cell cycle and shows that mitosis begins early in the division process and coincides approximately with the earliest stages of oral morphogenesis (about 0.6 in the cell cycle in synchronous cell samples). The cc1 mutation blocks cell cycle progression prior to the point of commitment to division. Although the cc1 mutation blocks macronuclear DNA synthesis under restrictive conditions, it does not block micronuclear DNA synthesis. However, absence of functional cc1 gene product leads to blockage of micronuclear mitosis prior to completion of anaphase. This point coincides with commitment to division and is also the point at which oral morphogenesis is blocked in cc1 cells. The tim-ings of the transition points for micronuclear mitosis and oral morphogenesis in cc1 cells are closely associated in both synchronous cell samples and in asynchronous cultures. © 1992 Wiley-Liss, Inc.     

ON THE EXISTENCE OF A Go-PHASE IN THE CELL CYCLE

The model is based on the assumption that the cell cycle contains a G_o-phase which cells leave randomly with a constant probability per unit time, γ. After leaving the G_o-phase, the cells enter the C-phase which ends with cell division. The C-phase and its constituent phases, the‘true’G₁-phase, the S-phase, the G₂-phase and mitosis are assumed to have constant durations of T, T₁T_s, T₂ and T_m, respectively. For renewal tissue it is assumed that the probability per unit time of being lost from the population is a constant for all cells irrespective of their position in the cycle. The labelled mitosis curve and labelling index for continuous labelling are derived in terms of γ, T, and T_s. The model generates labelled mitosis curves which damp quickly and reach a constant value of twice the initial labelling index, if the mean duration of the G_o-phase is sufficiently long. It is shown that the predicted labelled mitosis and continuous labelling curves agree reasonably well with the experimental curves for the hamster cheek pouch if T has a value of about 60 hr. Data are presented for the rat dorsal epidermis which support the assumption that there is a constant probability per unit time of a cell being released from the Go-phase.     

Analysis of gemini pollen 3 mutant suggests a broad function of AUGMIN in microtubule organization during sexual reproduction in Arabidopsis

In flowering plants, male gametes arise via meiosis of diploid pollen mother cells followed by two rounds of mitotic division. Haploid microspores undergo polar nuclear migration and asymmetric division at pollen mitosis I to segregate the male germline, followed by division of the germ cell to generate a pair of sperm cells. We previously reported two gemini pollen (gem) mutants that produced twin‐celled pollen arising from polarity and cytokinesis defects at pollen mitosis I in Arabidopsis. Here, we report an independent mutant, gem3, with a similar division phenotype and severe genetic transmission defects through pollen. Cytological analyses revealed that gem3 disrupts cell division during male meiosis, at pollen mitosis I and during female gametophyte development. We show that gem3 is a hypomorphic allele (aug6‐1) of AUGMIN subunit 6, encoding a conserved component in the augmin complex, which mediates microtubule (MT)‐dependent MT nucleation in acentrosomal cells. We show that MT arrays are disturbed in gem3/aug6‐1 during male meiosis and pollen mitosis I using fluorescent MT‐markers. Our results demonstrate a broad role for the augmin complex in MT organization during sexual reproduction, and highlight gem3/aug6‐1 mutants as a valuable tool for the investigation of augmin‐dependent MT nucleation and dynamics in plant cells.     

Ultraviolet Light-Induced Division Delay in Synchronized Chinese Hamster Cells

The age-dependent, ultraviolet light (UVL) (254 nm)-induced division delay of surviving and nonsurviving Chinese hamster cells was studied. The response was examined after UVL exposures adjusted to yield approximately the same survival levels at different stages of the cell cycle, 60% or 30% survival. Cells irradiated in the middle of S suffered the longest division delay, and cells exposed in mitosis or in G₁ had about the same smaller delay in division. Cells irradiated in G₂, however, were not delayed at either survival level. It was further established, after exposures that yielded about 30% survivors at various stages of the cycle, that surviving cells had shorter delays than nonsurvivors. This difference was not observed for cells in G₂ at the time of exposure; i.e., neither surviving nor nonsurviving G₂ cells were delayed in division. The examination of mitotic index vs. time revealed that most cells reach mitosis, but all of the increase in the number of cells in the population can be accounted for by the increase of the viable cell fraction. These observations suggest strongly that nonsurviving cells, although present during most of the experiment, are stopped at mitosis and do not divide. Cells in mitosis at the time of irradiation complete their division, and in the same length of time as unirradiated controls. Division and mitotic delays after UVL are relatively much larger than after X-ray doses that reduce survival to about the same level.     

Gas2l3, a Novel Constriction Site-Associated Protein Whose Regulation Is Mediated by the APC/CCdh1 Complex

Growth arrest-specific 2-like protein 3 (Gas2l3) was recently identified as an Actin/Tubulin cross-linker protein that regulates cytokinesis. Using cell-free systems from both frog eggs and human cells, we show that the Gas2l3 protein is targeted for ubiquitin-mediated proteolysis by the APC/C^Cdh1 complex, but not by the APC/C^Cdc20 complex, and is phosphorylated by Cdk1 in mitosis. Moreover, late in cytokinesis, Gas2l3 is exclusively localized to the constriction sites, which are the narrowest parts of the intercellular bridge connecting the two daughter cells. Overexpression of Gas2l3 specifically interferes with cell abscission, which is the final stage of cell division, when the cutting of the intercellular bridge at the constriction sites occurs. We therefore suggest that Gas2l3 is part of the cellular mechanism that terminates cell division.     

Expression of Bombyx mori 30Kc19 protein in Escherichia coli and its anti-apoptotic effect in Sf9 cell

The silkworm hemolymph has an anti-apoptotic activity in insect, mammalian, and human cell systems. The protein from silkworm hemolymph with the highest apoptosis inhibiting activity was found to be 30Kc19 protein, which was one of the ‘30K proteins’. In this study, 30Kc19 protein encoded by the 30Kc19 gene of the silkworm was expressed in Escherichia coli with (pET-22b(+)) and without (pET-3a) pelB leader sequence. 30Kc19 protein was over-expressed largely as a soluble form by pET-3a and both as soluble and insoluble forms by pET-22b(+). The medium was supplemented with each of the recombinant 30Kc19 proteins, and their presence was found to inhibit nuclear fragmentation and apoptotic body formation in actinomycin D-induced Sf9 cell apoptosis. Moreover, 30Kc19 protein repressed the activation of Sf-caspase-1. The 30Kc19 protein obtained from periplasm showed the most effective anti-apoptotic activity. This protein holds great potential for industrial and pharmaceutical applications since mass production and easy purification of this protein is possible.     

Ultrastructure of the Giant Amoeba Pelomyxa palustris*

SYNOPSIS. The ultrastructure of the herbivorous amoeba Pelomyxapalustris was studied. Nuclear division is not understood in this amoeba, and evidence for the method of nuclear division was sought. This species typically has many spheroidal nuclei which are similar within a given cell. However, some amoebae from our collections differed from this common type in both the number and structure of their nuclei. This suggested stages associated with nuclear division. One current hypothesis of nuclear division in this organism is that of nuclear budding. Our evidence is more in accord with this method than with mitosis. The cytoplasm contained no mitochondria, Golgi bodies, contractile vacuoles or crystals. Most amoebae had 2 types of bacteria (bacteroids or endosymbionts) in their cytoplasm; a separate vesicle enclosed each of these. Characteristically, only 1 type of bacterium (B_n) surrounded the nucleus. Another type (B) was found elsewhere in the cytoplasm. Also in the cytoplasm were the following: food vacuoles enclosing various algae, relatively clear vacuoles and vesicles, glycogen, various electron-opaque particles, and occasional microtubules. The plasmalemma was smooth, lacking the external fringe which characterizes other large fresh-water amoebae.     

Pre-prophase bands of microtubules in all categories of formative and proliferative cell division in Azolla roots

Pre-prophase bands of microtubules were found in every category of cell division, symmetrical and asymmetrical, in the cell lineages of the root apex of Azolla pinnata R.Br. and A. filiculoides Lam., and in the transverse divisions in the cell files of the roots. They are also found in the asymmetrical cell division that gives rise to trichoblasts in roots of Hydrocharis dubia (B1). Backer. It is possible, in a variety of cell types in roots of Azolla, to predict within a fraction of a micrometre where a new cell wall will be located. In every such case the midline of the 1.5–3-m-wide pre-prophase band anticipates this location. Each of the daughter cells thus inherits approximately half of the former pre-prophase band site. Images interpreted as stages of formation of the band were obtained, its microtubules replacing the interphase cortical arrays. In one highly asymmetrical division, band formation precedes migration of the nucleus to the site of mitosis. The asymmetrical division that gives rise to root hairs passes acropetally along every cell in the dermatogen layer, and preprophase bands were seen up to 8 cells in advance of the last completed division. Here, and in the zone of formative divisions, the band is present for much longer than the duration of mitosis. The ubiquity of the band in the Azolla root tip is discussed in relation to the literature, and a working hypothesis is presented that takes into account current knowledge of occurrence, development and function of the band.     

ULTRASTRUCTURE OF CELL DIVISION AND REPRODUCTIVE DIFFERENTIATION OF MALE PLANTS IN THE FLORIDEOPHYCEAE (RHODOPHYTA): CELL DIVISION IN POLYSIPHONIA1

Cell division in the marine red algae Polysiphonia harveyi Bailey and P. denudata (Dillwyn) Kutzing was studied with the electron microscope. Cells comprising the compact spermatangial branches of male plants were used exclusively because of their small size, large numbers and the ease with which the division planes can be predetermined. Some features characterizing mitosis in Polysiphonia confirm earlier electron microscope observations in Membranoptera, the only other florideophycean algae in which mitosis has been studied in detail. Common to both genera are a closed, fenestrated spindle, perinuclear endoplasmic reticulum, a typical metaphase plate arrangement of chromosomes, conspicuous, layered kinetochores, chromosomal and non-chromosomal microtubules, and nucleus associated organelles (NAOs) known as polar rings (PRs) located singly in large ribosome-free zones of exclusion at division poles in late prophase. However, other features, unreported in Membranoptera, were observed consistently in Polysiphonia. These include the presence of PR pairs in interphase-early prophase cells, the attachment of PRs to the nuclear envelope during all mitotic stages, the migration of a single PR to establish the division axis, a prominent, nuclear envelope protrusion (NEP) at both division poles at late prophase, the prometaphase splitting of PRs into proximal and distal portions, and the reformation of post-mitotic nuclei by the separation of an elongated interzonal nuclear midpiece at telophase. During cytokinesis, cleavage furrows impinge upon a central vacuolar region located between the two nuclei and eventually pit connections are formed in a manner basically similar to that reported for other red algae. Diagrammatic sequences of proposed PR behavior during mitosis are presented which can account for events known to occur during cell division in Polysiphonia. Mitosis is compared with that reported in several other lower plants and it is suggested that features of cell division are useful criteria to aid in the assessment of phylogenetic relationships of red algae.