Early Stages of Nematode-induced Giant-cell Formation in Roots of Impatiens balsamina

Giant cells induced in roots of Impatiens balsamina by Meloidogyne javanica and Meloidogyne incognita have been examined by light and electron microscopy. The first sign of giant-cell formation was division of cells surrounding a larva. Cell plate alignment appeared to proceed normally, but cytokinesis was unsuccessful and binucleate cells formed subsequently. No wall breakdown was evident then or later. The number of nuclei appeared to increase by repeated mitosis without separation by cytokinesis. Although no holes in walls were observed, wall stubs were found, and mechanisms for their formation are suggested.     

A non-genetic route to aneuploidy in human cancers

Aneuploidy is common in human tumours and is often indicative of aggressive disease. Aneuploidy can result from cytokinesis failure, which produces binucleate cells that generate aneuploid offspring with subsequent divisions. In cancers, disruption of cytokinesis is known to result from genetic perturbations to mitotic pathways or checkpoints. Here we describe a non-genetic mechanism of cytokinesis failure that occurs as a direct result of cell-in-cell formation by entosis. Live cells internalized by entosis, which can persist through the cell cycle of host cells, disrupt formation of the contractile ring during host cell division. As a result, cytokinesis frequently fails, generating binucleate cells that produce aneuploid cell lineages. In human breast tumours, multinucleation is associated with cell-in-cell structures. These data define a previously unknown mechanism of cytokinesis failure and aneuploid cell formation that operates in human cancers.     

Growth and development in relation to the cell cycle inPhysarum polycephalum

Summary In strain CL ofPhysarum polycephalum, multinucleate, haploid plasmodia form within clones of uninucleate, haploid amoebae. Analysis of plasmodium development, using time-lapse cinematography, shows that binucleate cells arise from uninucleate cells, by mitosis without cytokinesis. Either one or both daughter cells, from an apparently normal amoebal division, can enter an extended cell cycle (28.7 hours compared to the 11.8 hours for vegetative amoebae) that ends in the formation of a binucleate cell. This long cycle is accompanied by extra growth; cells that become binucleate are twice as big as amoebae at the time of mitosis. Nuclear size also increases during the extended cell cycle: flow cytometric analysis indicates that this is not associated with an increase over the haploid DNA content. During the extended cell cycle uninucleate cells lose the ability to transform into flagellated cells and also become irreversibly committed to plasmodium development. It is shown that commitment occurs a maximum of 13.5 hours before binucleate cell formation and that loss of ability to flagellate precedes commitment by 3–5 hours. Plasmodia develop from binucleate cells by cell fusions and synchronous mitoses without cytokinesis.Abbreviations CL Colonia Leicester - DSDM Dilute semi-defined medium - FKB Formalin killed bacterial suspension - IMT Intermitotic time - LIA Liver infusion agar - SBS Standard bacterial suspension - SDM Semi-defined medium     

Analysis of development and growth in a mutant of Physarum polycephalum with defective cytokinesis

Cultures of amoebae of the mutant strain ATS23 isolated from strain CLd of Physarum polycephalum contain multinucleate cells and cells with increased nuclear DNA content. Plasmodia derived from ATS23 clones show abnormal morphology and defective sporulation. All abnormalities are enhanced by high incubation temperature (31 °C). Genetic analysis suggested that all the abnormalities were caused by a single mutation, denoted hts-23. The kinetics of plasmodium formation were followed in cultures of apogamic amoebae carrying hts-23 and hts⁺ (wild type) respectively. Results indicated that, relative to wild type, hts-23 did not increase the rate of plasmodium formation. There was evidence that, in both mutant and wild-type strains, commitment to plasmodium development occurred in uninucleate cells. Analysis of cell pedigrees by time-lapse cinematography indicated that the primary abnormal event in cultures of hts-23 amoebae was failure of cytokinesis; an apparently complete cleavage furrow was formed but cell separation failed, resulting in a binucleate cell. This event occurred randomly in pedigrees in which the majority of divisions were completed normally; its frequency increased during incubation at 31 °C. All other abnormalities in hts-23 amoebal cultures could be attributed to this primary event, assuming that DNA synthesis continued in the absence of cytokinesis and that the binucleate cells underwent the amoebal type of “open” mitosis, allowing the possibility of spindle fusion. This implies that the acquisition of “closed” mitosis is an essential early step in plasmodium development.     

Contribution to the Understanding of the Mechanism of Cytokinesis In Plant Cells: the Action of Deoxyguanosine On the Kinetics of A Root Meristem Cell Population

The kinetics of binucleate cells, formed by the action of deoxyguanosine, are studied using three methods: in a population synchronized with hydroxyurea, by autoradiography after pulse-labelling, and in a sample of a cell population morphologically located at the M-G₁, limit. Deoxyguanosine induces a slowing down in S and G₂, independent of the inhibition of cytokinesis. It is only when it takes effect during the G₂, stage that deoxyguanosine brings about the formation of binucleate cells.     

Contribution to the understanding of the mechanism of cytokinesis in plant cells: the action of deoxyguanosine on the kinetics of a root meristem cell population.

The kinetics of binucleate cells, formed by the action of deoxyguanosine, are studied using three methods: in a population synchronized with hydroxyurea, by autoradiography after pulse-labelling, and in a sample of a cell population morphologically located at the M--G1 limit. Deoxyguanosine induces a slowing down in S and G2, independent of the inhibition of cytokinesis. It is only when it takes effect during the G2 stage that deoxyguanosine brings about the formation of binucleate cells.     

Heterovesicula cowani N. G., N. Sp. (Heterovesiculidae N. Fam.), a Microsporidian Parasite of Mormon Crickets, Anabrus simplex Haldeman, 1852 (Orthoptera: Tettigoniidae)

ABSTRACT. Heterovesicula cowani , n. g., n. sp., is a dimorphic microsporidium described from the adipose tissue of the Mormon cricket, Anabrus simplex Haldeman. Proliferation of the microsporidium is by karyokinesis of uninucleate and binucleate cells to form binucleate and tetranucleate cells, respectively. These cells will undergo binary fission (merogony). Ultimately, the meronts undergo karyokinesis without subsequent cytokinesis producing spherical multinucleate plasmodia that are transitional to 2 types of sporogony. Transitional to disporoblastic sporogony, a fragile interfacial envelope delaminates from the plasmodium with morphogenesis to a monfiliform plasmodium consisting of fusiform binucleate diplokaryotic sporonts. These undergo karyokinesis to form tetranucleate diplokaryotic sporonts that undergo cytokinesis during disintegration of the plasmodium into isolated binucleate sporonts. Transitional to octosporoblastic sporogony, multinucleate plasmodia disintegrate into short monofiliform plasmodia of diplokaryotic sporonts which then segregate while undergoing gradual nuclear dissociation (haplosis by nuclear dissociation). These undergo two sequences of karyokinesis and subsequent multiple fission to form eight uninucleate (haploid) sporoblasts in a fusiform arrangement within a persistent envelope. Binucleate spores are ovocylindrical, about 5.4 × 1.7 μm (fresh), with an isofilar polar filament singly coiled about 11 turns. Uninucleate spores are ovoid to slightly pyriform, 4.0 × 1.7 μm (fresh), with an isofilar filament singly coiled about 9 turns. A new family, Heterovesculidae, is proposed for the new genus.     

Cell division in caffeine-induced binucleate protonemal cells ofAdiantum. II. Formation of the preprophase band and cell division in centrifuged and non-centrifuged cells

Organization of microtubules (MTs) in relation to the behavior of nuclei was examined in dividing binucleate cells ofAdiantum capillus-veneris L. To induce binucleate cells, caffeine, an inhibitor of formation of the cell plate, was applied at 4 mM to synchronously dividing protonemal cells during cytokinesis (Murata and Wada 1993). Formation of the preprophase band (PPB) during the next cell cycle was examined in non-centrifuged and centrifuged cells. The two nuclei were separated or associated with one another in both non-centrifuged and centrifuged cells, although the location of the nuclei in the cylindrical protonemal cells was different (Murata and Wada 1993). Irrespective of centrifugation, a single PPB was formed around the nuclei in cells with associated nuclei. Two PPBs were formed in cells with separated nuclei in centrifuged cells. Patterns of mitosis and cytokinesis varied, depending on the location of the PPB and the distribution of the nuclei. The role of the nucleus in formation of the PPB is discussed.     

Inhibition of cytokinesis by asbestos and synthetic fibres

Using high-resolution timelapse microscopy, we have followed individual phagocytized fibres through the later stages of division in MeT-5A human mesothelial cells and LLC-MK(2)monkey epithelial cells. The fibres used were crocidolite and chrysotile asbestos, fibrous glass (MMVF), and refractory ceramic fibres (RCF). Long fibres (15-80 microm) trapped within the cleavage furrow can partially or completely block cytokinesis. Cells proceed in one of three ways: (1) eventual completion of cytokinesis; (2) incomplete cytokinesis, resulting in two cells joined by a fibre-containing intercellular channel; or (3) failure of cytokinesis, resulting in a binucleate or trinucleate cell. Two factors associated with fibre-induced bi/trinucleation are: (1) an initial association between the fibre and the forming daughter nuclei, which is sometimes lost over time, and (2) disintegration of the midbody. The studies suggest that delay of cytokinesis by interzonal fibres can result in bi/trinucleation through the loss of midbody/intercellular bridge proteins that are required for completion of cytokinesis.     

Variable pathways for developmental changes of mitosis and cytokinesis in Physarum polycephalum

The development of a uninucleate ameba into a multinucleate, syncytial plasmodium in myxomycetes involves a change from the open, astral mitosis of the ameba to the intranuclear, anastral mitosis of the plasmodium, and the omission of cytokinesis from the cell cycle. We describe immunofluorescence microscopic studies of the amebal-plasmodial transition (APT) in Physarum polycephalum. We demonstrate that the reorganization of mitotic spindles commences in uninucleate cells after commitment to plasmodium formation, is completed by the binucleate stage, and occurs via different routes in individual developing cells. Most uninucleate developing cells formed mitotic spindles characteristic either of amebae or of plasmodia. However, chimeric mitotic figures exhibiting features of both amebal and plasmodial mitoses, and a novel star microtubular array were also observed. The loss of the ameba-specific alpha 3-tubulin and the accumulation of the plasmodium-specific beta 2-tubulin isotypes during development were not sufficient to explain the changes in the organization of mitotic spindles. The majority of uninucleate developing cells undergoing astral mitoses (amebal and chimeric) exhibited cytokinetic furrows, whereas cells with the anastral plasmodial mitosis exhibited no furrows. Thus, the transition from astral to anastral mitosis during the APT could be sufficient for the omission of cytokinesis from the cell cycle. However, astral mitosis may not ensure cytokinesis: some cells undergoing amebal or chimeric mitosis contained unilateral cytokinetic furrows or no furrow at all. These cells would, most probably, fail to divide. We suggest that a uninucleate committed cell undergoing amebal or chimeric mitosis can either divide or else form a binucleate cell. In contrast, a uninucleate cell with a mitotic spindle of the plasmodial type gives rise only to a binucleate cells. Further, the decision to enter mitosis after commitment to the APT is independent of the developmental changes in the organization of the mitotic spindle and cytokinesis.     

G2 arrest, binucleation, and single-parameter DNA flow cytometric analysis

One important facet of flow cytometry involves the effects of pharmacological agents on cell cycle progression. Comparative G2 fraction perturbations were examined: effects of sodium butyrate on articular chondrocytes, effects of an antineoplastic agent (SOAZ) and an antirheumatic drug (D-penicillamine) on HeLa cells. Even though DNA flow cytometric analysis detects preferentially an induction of G2 arrest, the mode of action of these agents on the cell cycle is different. Sodium butyrate and D-penicillamine lead to an increase of binucleate cells due to cytokinesis perturbation. Because of similar fluorescence intensity, distinguishing G2 from binucleate GO/1 cells is not easily possible using DNA content measurement and reflects a failure of flow cytometry in the detection of binucleate cells. Rapid cell cycle analysis of single cells should contribute greatly to the study of pharmacological interactions, but DNA flow cytometric measurements obtained from cultured cells exposed to certain agents must be cautiously interpreted because those may interact on cytokinesis and induce artefacts in histogram interpretation.     

Mechanism of ploidy shift from diploidy to tetraploidy in MSPC-1 mouse myeloma

In order to elucidate the cytological mechanism of ploidy shift from diploidy to tetraploidy in MSPC-1 mouse myeloma, the process of cell division was observed in living cells under phase contrast microscope. It was suggested that loss of cytokinesis and subsequent formation of binucleate cells are the major causes of such a ploidy shift. Elevated frequencies of binucleate cells during the transition phase of ploidy shift from diploidy to tetraploidy also supported the above notion. The possibility of cell fusion as a cause of the ploidy change could be eliminated by analyses of marker chromosomes and incorporation pattern of [³H]thymidine into binuclei.     

Bistratene A induces a microtubule-dependent block in cytokinesis and altered stathmin expression in HL60 cells.

Bistratene A is a cyclic polyether which affects cell cycle progression and can induce phosphorylation of cellular proteins. Treatment of HL60 cells with 100 ng/ml bistratene A was found to inhibit cytokinesis but had no effect on DNA synthesis and nuclear division. Consequently, bistratene A-treated cells became polyploid and multinucleate. In association with the development of this phenotype, the cytoplasmic protein stathmin was biphasically phosphorylated and levels of expression were doubled. Immunostaining of binucleate cells (bistratene A for 24 h) revealed increased alpha-tubulin localization where the cleavage furrow might be expected to form, i.e., along the equatorial plane. Treatment of these binucleate cells with the microtubule depolymerizing agent nocadazole promoted cleavage furrow formation and partially ameliorated the bistratene A-induced block in cell division. These findings implicate the polymerization status of microtubules and stathmin function in the regulation of cytokinesis.     

Comparative effects of caffeine, its analogues and calcium deficiency on cytokinesis

The effects of caffeine, aminophylline, caffeic acid, and calcium deficiency on cytokinesis were studied by light and electron microscopy. All these treatments blocked cell plate formation, resulting in the formation of binucleate cells. The aggregation and organization of membranous vesicles at the ‘presumptive cell plate’ during these treatments appears similar to that of normal cells, but fusion of the vesicles is insufficient to form a complete cell plate. It is suggested that some aspect of membrane recognition and fusion is the process actually interfered with by these treatments. Greater numbers of binucleate cells and fewer partial cell plates were observed in cells treated with caffeine and aminophylline as compared with those exposed to caffeic acid or calcium deficiency, indicating that the latter treatments do not block cell plate formation as efficiently as the former.     

Cell division in caffeine-induced binucleate protonemal cells ofAdiantum. I. Asynchronous mitosis of two nuclei in a single cell

Coordination of karyokinesis of two nuclei in individual filamentous binucleate cells of the fern,Adiantum capillus-veneris was investigated. To induce binucleate cells, the protonemata were treated with caffeine, which is known as an inhibitor of plant cytokinesis, during the first synchronous division of cells that was induced by blue light (BL). The next synchronous division of cells in the resultant binucleate cells was analysed. In most cases, the two nuclei were associated with each other and were located in the apical region of the long protonemal cells (approximately 400–600 μm in length, 20 μm in width). In some cells, one nucleus was located in the apical region and the other was located in the middle of the cylinderical region. In such cells, karyokinesis of the apical nucleus preceded that of the basal nucleus, even though karyokinesis of associated nuclei progressed synchronously. Mitotic binucleate cells were centrifuged in order to gather two dissociated heterophasic nuclei. Progression of karyokinesis in the re-associated nuclei became coordinated within 1 h in most cells. These results suggest that mitosis-regulating factor(s) may diffuse to only limited distances inAdiantum protonemata.     

Life cycle of a new species of Amblyospora (Microspora: Amblyosporidae) in the mosquito Aedes taeniorhynchus

A new species of Microspora, Amblyospora polykarya, is described from the mosquito Aedes taeniorhynchus. The parasite is transovarially transmitted for one generation only. Spores in adult females extrude binucleate sporoplasms which infect developing eggs. Merogony occurs in larval oenocytes with diplokaryotic stages in early instars giving rise to plasmodia with many diplokarya. Plasmodia undergo cytokinesis to form diplokaryotic sporonts. In fat body cells, these sporonts secrete pansporoblastic membranes and undergo two nuclear divisions to form octonucleate sporonts. Cytokinesis and differentiation result in uninucleate spores in packets of eight. These spores are not transmissible per os and are of different morphotype from those in adult females. Infected larvae die in the fourth stadium.     

The mode of nuclear DNA synthesis in experimentally induced binucleate cells of root meristems

The synthesis of DNA does not proceed in a perfectly synchronous way in about 20% of the binucleate cells induced in Allium sativum L. root meristems by treatment with methyl 3 hydroxy 6 (1 H, 3 H) quinazoline dione, 2.4 (an inhibitor of cytokinesis). — Nuclear labelling after tritiated thymidine incorporation and the measurement of nuclear DNA by cytophotometry both establish that the beginning of the S phase as well as its duration may be different in the two nuclei included in a single cytoplasm. Yet the nuclear synchronism progressively asserts itself as the cell cycle goes on and, following this asynchronous synthesis, the beginning of mitosis is synchronous. The restoration of such a coordination points to the existence in the cytoplasm of separable factors inducing DNA synthesis and mitosis.     

Triggering p53 after cytokinesis failure

Cells that fail to divide during cytokinesis often arrest in the next G1 phase by a mysterious mechanism that depends upon p53. What triggers this arrest is unclear. New studies, including a report in this issue (Uetake and Sluder, 2004) suggest that this arrest does not occur because cells are polyploid, are binucleate, have multiple centrosome, or have failed cytokinesis, making this phenomenon even more puzzling.     

Cell line which is temperature-sensitive for cytokinesis

We have isolated several cell lines which are temperature-sensitive for growth. One of these appears to be temperature-sensitive for cytokinesis. It was isolated from a Syrian hamster cell line by selecting cells which were not killed by 1 μg/ml cytosine arabinoside at 39° but which grew normally at 31°. It shows an increased proportion of binucleate cells when shifted to the non-permissive temperature and time-lapse photomicroscopy shows that a high proportion of attempted mitoses fail at 39°, apparently at the stage of cytokinesis. The cells which have failed to complete mitosis reattach to the plate and have two normal-size nuclei but otherwise behave normally.     

Rab11-FIP3 is a cell cycle-regulated phosphoprotein

Background

Rab11 and its effector molecule, Rab11-FIP3 (FIP3), associate with recycling endosomes and traffic into the furrow and midbody of cells during cytokinesis. FIP3 also controls recycling endosome distribution during interphase. Here, we examine whether phosphorylation of FIP3 is involved in these activities.

Results

We identify four sites of phosphorylation of FIP3 in vivo, S-102, S-280, S-347 and S-450 and identify S-102 as a target for Cdk1-cyclin B in vitro. Of these, we show that S-102 is phosphorylated in metaphase and is dephosphorylated as cells enter telophase. Over-expression of FIP3-S102D increased the frequency of binucleate cells consistent with a role for this phospho-acceptor site in cytokinesis. Mutation of S-280, S-347 or S-450 or other previously identified phospho-acceptor sites (S-488, S-538, S-647 and S-648) was without effect on binucleate cell formation and did not modulate the distribution of FIP3 during the cell cycle. In an attempt to identify a functional role for FIP3 phosphorylation, we report that the change in FIP3 distribution from cytosolic to membrane-associated observed during progression from anaphase to telophase is accompanied by a concomitant dephosphorylation of FIP3. However, the phospho-acceptor sites identified here did not control this change in distribution.

Conclusions

Our data thus identify FIP3 as a cell cycle regulated phosphoprotein and suggest dephosphorylation of FIP3 accompanies its translocation from the cytosol to membranes during telophase. S102 is dephosphorylated during telophase; mutation of S102 exerts a modest effect on cytokinesis. Finally, we show that de/phosphorylation of the phospho-acceptor sites identified here (S-102, S-280, S-347 and S-450) is not required for the spatial control of recycling endosome distribution or function.