Caffeine inhibition of cytokinesis: Ultrastructure of cell plate formation/degradation

Summary Caffeine is a potent inhibitor of cell plate formation in dividing plant cells. Previous studies living cells reveal that the drug always permits the cell plate to arise and grow normally until about 80% complete, but then causes it to break down. In the present investigation we examine this formation/degradation cycle at the ultrastructure level. Our results show that during the formation phase the caffeine treated plate is indistinguishable from untreated controls. Phragmoplast microtubules arise and align in the interzone, Golgi vesicles are produced and aggregate in a line that defines the young cell plate, and considerable fusion of these vesicles occurs to form islands of plate material. However, under the influence of caffeine these islands do not fuse to form the enlarged lamellar expanses characteristic of maturing cell plates. Instead, the partially fused material reverts to small vesicles which appear to become resorbed by the cellular membrane systems. The resorption process continues leaving no evidence of the previously developing plate, although occasionally we observe a stub of fused vesicles attached to the parent wall. Following cell plate disintegration the reformed nuclei move close together and occupy the central region of the cell. These observations focus attention on the consolidation phase of cell plate formation as the one being maximally affected by caffeine.Dedicated to the memory of Professor Oswald Kiermayer     

Caffeine inhibition of cytokinesis: effect on the phragmoplast cytoskeleton in livingTradescantia stamen hair cells

Summary The distribution of microtubules and actin microfilaments during caffeine-induced inhibition of cell plate formation has been studied in livingTradescantia stamen hair cells. Previous studies have shown that caffeine allows cell plate initiation but prevents its completion, resulting in binucleate cells. In the present study, confocal microscopy of cells microinjected with fluorescent brain tubulin or phalloidin, and cultured in the presence 5 mM caffeine, revealed that the initiation and early lateral expansion phase of the phragmoplast occur normally. However, caffeine completely inhibits the formation of the cytoskeletal torus which occurs in untreated cells during the late stages of cell plate and phragmoplast expansion. Caffeine further causes the disintegration of the incomplete cell plate. The results allow us to distinguish two phases in cell plate and phragmoplast growth: the initiation and early expansion phase, which is not affected by caffeine, and the late lateral expansion phase, which is completely inhibited in the presence of caffeine. Also in this study, the use of a high phalloidin concentration has revealed structural detail about the actin microfilaments involved in cell plate formation: microfilaments are observed that link the expanding edge of the phragmoplast with the cortical division site. In addition, cortical actin patches are observed within the actin depleted zone that might play a role in guidance of phragmoplast and cell plate expansion.     

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.     

Lithium induces cell plate dispersion during cytokinesis inTradescantia

Summary We have found that 10mM LiCl added toTradescantia stamen hair cells prior to early anaphase appears to prevent the vesicle coalescence phase of cell plate formation. In a fashion similar to caffeine inhibition of cell plate formation [Bonsignore and Hepler 1985, Protoplasma 129, 28–35], cell plate vesicle aggregation occurs at its normal time in LiCl, forming an incipient plate which is similar in morphology to that of an untreated cell during the first 10min, but the structure subsequently disperses and the resultant cells are binucleate. The addition of 10–20M myo-inositol was sufficient to reverse the inhibitory effect of Li⁺ in the majority of our experiments while scyllitol, an isomer of myo-inositol, or buffer without myoinositol were usually insufficient to reverse the inhibition. The timing of the addition of myo-inositol was critical for reversal; if the rescue solution was added more than 4 min after the onset of cell plate vesicle aggregation, the cell was usually irreversibly destined to become binucleate. The addition of 100M CaCl₂ within 2min of cell plate vesicle aggregation also overcame Li⁺-induced plate dispersal, but the kinetics of reversal were substantially slower than with myo-inositol. The results show that cell plate formation and in particular, cell plate vesicle coalescence, is sensitive to exogenously applied LiCl.Abbreviations AO Anaphase Onset - CP Cell Plate (completed) - CPD Cell Plate Dispersion - CPVA Cell Plate Vesicle Aggregation - DAG 1,2-diacylglycerol - InsP₃ 1,4,5-inositol trisphosphate - IP inositol-1-phosphate - polyPI polyphosphoinositide     

Rotokinesis, a novel phenomenon of cell locomotion-assisted cytokinesis in the ciliate Tetrahymena thermophila

The mechanism responsible for final cell separation at the end of cytokinesis is currently unknown. Knockout strains of the ciliate, Tetrahymena thermophila lacking the kinesin-II homologous molecular motors, Kin1p and Kin2p are paralyzed due to their complete loss of cilia and undergo frequent cytokinesis failures. Observations of live dividing cells revealed that cleavage furrow ingression is normal in kinesin-II double knockout cells until the final stage of cell separation (Brown et al., 1999). During closer inspection of dividing cells using video differential interference contrast microscopy, we found that wild-type cells undergo an extremely complex motile behavior near the end of cytokinesis. This process, which we have named rotokinesis, appears to facilitate the physical separation of daughter cells. Here we present recent work onTetrahymena rotokinesis, and review studies in other organisms which suggest that the use of cell locomotion in the completion of cytokinesis is a general phenomenon of motile cell types.     

A centrin homologue is localised across the developing cell plate in gymnosperms and angiosperms

Summary A homologue of centrin, a calcium-binding protein, has been found in some land plants and shown by immunochemistry to localise prominently to the cell plate in angiosperms. In the present study, we used immunochemistry to extend these observations to gymnosperms and to further our understanding of centrin localisation in the two divisions. In Monterey pine, immunoblotting revealed an 18 kDa centrin homologue. Immunofluorescence confocal microscopy of root-tip cells of pine and onion and three-dimensional reconstruction showed that a centrin homologue is localised across the developing cell plate. The localisation extended both to the zone of overlap of the two interdigitating sets of phragmoplast microtubules at the edge of the expanding cell plate and to the remainder of the plate devoid of phragmoplast microtubules. Induction of cytokinetic arrest in onion andArabidopsis thaliana by caffeine or brefeldin A produced disrupted phragmoplasts and centrin-labelled cell plates, indicating that the localisation of centrin is coupled to the deposition of the cell plate by the phragmoplast.     

Dynamics of phragmoplastin in living cells during cell plate formation and uncoupling of cell elongation from the plane of cell division.

The cell plate is formed by the fusion of Golgi apparatus-derived vesicles in the center of the phragmoplast during cytokinesis in plant cells. A dynamin-like protein, phragmoplastin, has been isolated and shown to be associated with cell plate formation in soybean by using immunocytochemistry. In this article, we demonstrate that similar to dynamin, phragmoplastin polymerizes to form oligomers. We fused soybean phragmoplastin with the green fluorescence protein (GFP) and introduced it into tobacco BY-2 cells to monitor the dynamics of early events in cell plate formation. We demonstrate that the chimeric protein is functional and targeted to the cell plate during cytokinesis in transgenic cells. GFP-phragmoplastin was found to appear first in the center of the forming cell plate, and as the cell plate grew outward, it redistributed to the growing margins of the cell plate. The redistribution of phragmoplastin may require microtubule reorganization because the microtubule-stabilizing drug taxol inhibited phragmoplastin redistribution. Our data show that throughout the entire process of cytokinesis, phragmoplastin is concentrated in the area in which membrane fusion is active, suggesting that phragmoplastin participates in an early membrane fusion event during cell plate formation. Based on the dynamics of GFP-phragmoplastin, it appears that the process of cell plate formation is completed in two phases. The first phase is confined to the cylinder of the phragmoplast proper and is followed by a second phase that deposits phragmoplast vesicles in a concentric fashion, resulting in a ring of fluorescence, with the concentration of vesicles being higher at the periphery. In addition, overexpression of GFP-phragmoplastin appears to act as a dominant negative, slowing down the completion of cell plate formation, and often results in an oblique cell plate. The latter appears to uncouple cell elongation from the plane of cell division, forming twisted and elongated cells with longitudinal cell divisions.     

Studies on formation and resorption of fish scales

Summary Scale formation in Cyprinodon variegatus was found to be initiated at about 26 to 30 days after hatching. Ultrastructural investigation revealed that within 4 to 6 h in the first-formed scales the marginal cells begin to flatten and differentiate into osteogenic cells, which later change to osteoblasts and fibroblasts. These cells are separated from the surrounding epithelial cells by a basal lamina. The osteoid is formed by the marginal and osteogenic cells; the osseous layer by the osteoblasts; and the fibrillary plate by the fibroblasts.The osteoid is formed within 2 to 3 h after the initiation of the scale, and within 20 to 24 h the osseous layer is formed. Hydroxyapatite crystals are deposited in the matrix of the osseous layer without apparent association with collagen fibers. No matrix vesicles or dense bodies are evident at the sites of calcification. The fibrillary plate arises 18 to 20 h after the initiation of the scale. It is also partially calcified, but not before the third week of scale formation. The crystals develop almost exclusively between the collagen fibers at the extreme edge of the calcifying front, but solid calcification of the fibers results with further growth of the crystals. The fibroblasts appear to participate in calcification of the fibrillary plate.Contribution No. 332, Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina, 29208, USA     

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.     

cyd,a mutant of pea that alters embryo morphology is defective in cytokinesis

Embryogenesis in higher plants requires the precise regulation of cell division, orientation of cell elongation and specification of cell differentiation. The division plane is determined by the position of a new cell plate at cytokinesis. A mutant of pea has been isolated in which both the embryo pattern and surface morphology is altered. The phenotype of the mutant is manifest primarily in the cotyledons where cell plates only partially form, generating cell wall stubs and multinucleate cells. Some cotyledonary cells of the mutant proceed through nine DNA replication cycles, including nuclear division, but not cytokinesis, producing nuclei with a DNA content of ca. 1000C. The cytological phenotype of the mutant could be mimicked by the treatment of wild-type cells with caffeine. We have termed this mutant cytokinesis-defective (cyd). © 1995 Wiley-Liss, Inc.     

Sélection,par les chromosomes en écouvillon,de lignées vectrices de mutations chez l'amphibien urodèle Pleurodeles poireti

Division of the guard mother cell (GMC) in Allium cotyledons has been examined in epidermal slices viewed with Nomarski optics and electron microscopy. Special attention has been directed towards elucidating the process by which the dividing cell determines its plane of division. In normal development, the cell plate formed during GMC division ultimately lies along the longitudinal axis of the cotyledon, in contrast to the transverse planes formed in other epidermal divisions. Our observations reveal that the final plane of division is not determined by the orientation of the spindle at metaphase but instead is established during late anaphase-telophase as a result of directed reorientation movements of the spindle-phragmoplast and associated daughter nuclei. The metaphase plate may lie at an oblique angle, even as great as 90°, from the final plane of the plate. Thus, daughter chromosomes separate into opposite corners of the cell. During late anaphase-telophase, movement of the spindle is activated; the daughter nuclei move along the sides of the cell while the interzone rotates. Movement continues until daughter nuclei reach positions opposite each other along the sides of the cell and the midzone or cell plate is positioned in the longitudinal orientation. Movement requires 15–20 minutes for completion, is highly directional, and does not overshoot the correct alignment. Following movement cytokinesis proceeds to completion forming two young guard cells. Possible mechanisms for reorientation are discussed, including one that suggests that interzone microtubules may interact with a cortical site on the plasmalemma adjacent to the end and paradermal walls. Such a site may be related to and governed by the same properties which controlled the prior formation of the preprophase band of microtubules in these cells.     

Centripetal wall formation in roots ofVicia faba after caffeine treatment

Summary Lateral roots ofVicia faba were treated three hours with 0.2 percent caffeine. The ultrastructure of binucleate cells formed that way was studied by electron microscopy. Shortly after the end of treatment, nuclei connected by a strand of nucleoplasm were observed which was referred to nuclear fusions. In binucleate cells no stages reminding of a typical phragmoplast or a cell plate could be identified, whereas wall protrusions occurred at interphase and mitosis, respectively, obviously growing centripetally performing a pseudo-cytokinesis. Some hours later wall formation was more irregular and nuclear constrictions could be observed. Since the microtubules are not affected, the possible effect of caffeine on cytoplasmic streams is discussed.     

The effect of adenosine on caffeine inhibition of plant cytokinesis

Adenosine counteracts caffeine-induced cytokenesis inhibition in meristem cells of onion root tips; counteracts caffeine-induced cytokinesis inhibition in meristen cells of onion root tips; dinitrophenol (DNP) potentiates this caffeine effect. These effects suggest that caffeine could act as a negative, and some adenosine derivatives as a positive, effector on some enzyme(s) essentially involved in cytokinesis. We postulate that caffeine can block cell plate formation by inhibition of a certain ATPase activity essential for membrane fusion of Golgi vesicles. A general mechanism which could explain many of the biological effects of methyloxypurines is proposed.     

Relationship of chromosomal damage induced by caffeine to growth temperature and ATP level in proliferating cells

Caffeine is known to induce chromosomal aberrations in proliferating cells when they are incubated during G2 and mitotic prophase. In the present paper, this caffeine effect has been analyzed in Allium cepa root meristems growing at different culture temperatures under steady-state kinetics. Caffeine (1-10 mM) induces chromosomal aberrations in a dose-dependent manner, and the treatment efficiency correlates linearly with the square of caffeine concentration. The efficiency of caffeine incubations, within the range 5-25 degrees C during equivalent cycle time periods has also been studied. It has been found that the lower the culture temperature, the higher the level of chromosomal aberrations. Moreover, at different temperatures, the level of chromosomal aberrations is a simple function of caffeine concentration and the ATP level. Therefore, the efficiency of caffeine treatment appears to be determined by some interaction between caffeine concentration and cellular ATP level. Our present results demonstrate that the influence of growth temperature on the chromosome-breaking effect of caffeine can be, at least partially, explained by the ATP levels during the incubation periods. In short, under different kinetics of plant cell proliferation, the ATP level, and/or something correlating with it, could explain the efficiency of caffeine in inducing chromosomal aberrations: the lower the ATP level, the higher the caffeine efficiency.     

Origin of sperm cell association in the “male germ unit” ofBrassica pollen

The association of the two sperm cells inBrassica napus pollen following the generative cell division was investigated. The generative cell during division is located in the center of the pollen grain, within the vegetative cell. The space present between the two cells is slightly irregular as seen following standard glutaraldehyde fixation. After completion of mitosis vesicles appear in the equatorial plane, coalescing centripetally to form a cell plate which fuses with the membrane of the generative cell, dividing it in two sperm cells. They are isolated from the vegetative cell by the space between the two cell membranes and are separated from each other by a similar space resulting from the cell plate formed during cytokinesis.     

Distribution of the Golgi apparatus in the mitosis of cultured tobacco cells as revealed by DiOC6 fluorescence microscopy

Summary We developed a new method for distinguishing the Golgi apparatus from the other membranous organelles which contain DNA, such as mitochondria and chloroplasts, under a fluorescence microscope. Thin sections of cells embedded in Technovit 8100 resin were stained with both 3,3-dihexyloxacarbocyanine iodide (DiOC₆) and 4,6-diamidino-3-phenylindole (DAPI), and those three membranous organelles were observed under an epifluorescence microscope. The Golgi apparatus, which do not contain DNA, were easily recognized when the two images stained with DiOC₆ and DAPI were superimposed using an image processor. Using this method, we investigated the dynamics of cellular membranes and organelles during the mitotic cycle of synchronized cultured tobacco cells BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2). The Golgi apparatus did not accumulate in the rim of the formating early cell plate at anaphase, while it accumulated near the maturing cell plate at telophase, and this accumulation seemed to be related to the maturation of cell plates. To confirm this hypothesis, synchronized BY-2 cells were treated with caffeine, which is known to inhibit the cell plate formation. Most of the cells treated with caffeine remained in a phase in which Golgi vesicles were accumulated at the equatorial plate, but the cell plate was only partially maturing. The Golgi apparatus accumulated only near the partially maturing cell plate, but not by the equatorial plate where the Golgi vesicles had accumulated.Abbreviations DiOC₆ 3,3-dihexyloxacarbocyanine iodide - DAPI 4,6-diamidino-3-phenylindole - LSD a modified Linsmaier and Skoog's medium containing 2,4-D     

Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe

Summary Twenty seven recessive temperature sensitive mutants have been isolated in Schizosaccharomyces pombe which are unable to complete the cell division cycle at the restrictive temperature. These mutants define 14 unlinked genes which are involved in DNA synthesis, nuclear division and cell plate formation. The products from most of these genes complete their function just before the cell cycle event in which they are involved. Physiological characterisation of the mutants has shown that DNA synthesis and nuclear division form a cycle of mutually dependent events which can operate in the absence of cell plate formation. Cell plate formation itself is usually dependent upon the completion of nuclear division.     

New 3-substituted xanthines : Potent inhibitors of cell plate formation

Caffeine and some related compounds are known to inhibit cell plate formation (CPF), but little is known about their mechanism of action. In this connection, the effects of xanthine derivatives on CPF in Tradescantia stamen hair cells were examined. Caffeine (1,3,7-trimethylxanthine) and 1,3-dimethyl-xanthine were more effective inhibitors of CPF than 1-methyl- or 1,7-dimethyl-xanthine. Four compounds with a substituent at the three position of 1,7-dimethyl-xanthine were found to be more effective than caffeine as inhibitors of CPF. Among them, 1,7-dimethyl-3-(3-ethylbutyl) xanthine was the most effective and the concentration required for inhibition of CBF was one tenth of that of caffeine. Breakdown of cell plates could be partially reversed using guanosine or adenosine, but reversibility was not detected using cytokinins.     

Genetic dissection of cytokinesis

Higher plants have evolved specific mechanisms for partitioning the cytoplasm of dividing cells. In the predominant mode of phragmoplast-assisted cytokinesis, a cell wall and flanking plasma membranes are made de novo from a transient membrane compartment, the cell plate, which in turn forms by vesicle fusion from the centre to the periphery of the dividing cell. Other modes of cytokinesis appear to occur in meiotic cells and developing gametophytes. Here we review recent progress in the analysis of plant cytokinesis, focusing on genetic studies in Arabidopsis which are beginning to identify structural and regulatory components of phragmoplast-assisted cytokinesis. Two classes of mutations have been described. In one class, the defects appear to be confined to cell plate formation, suggesting that the execution of cytokinesis is specifically affected. Mutations in the other class display more general defects in cell division. We also discuss possible roles of proteins that have been localised in cytokinetic cells but not characterised genetically. Finally, mutations affecting meiotic or gametophytic cell divisions suggest that mechanistically different modes of cytokinesis occur in higher plants.     

Regulation of polar cap formation in the life cycle of Escherichia coli

Polar cap formation has been studied in synchronized Escherichia coli cells. It is dependent on a signal given after completion of a round of DNA replication. A 20 min time interval between the release of this signal and physical cell separation is probably the time required for the completion of polar caps. During this time murein is synthesized at an increased rate and cells are especially sensitive to penicillin.