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     

Inhibition of cyclic GMP formation and aggregation in Dictyostelium by the intracellular Ca2+ antagonist TMB-8

Aggregation in Dictyostelium discoideum was shown in previous studies employing EGTA to require Ca2+, but the intra- or extracellular site of action of this ion and its role in chemotaxis were not determined [1]. In this investigation we show that the intracellular Ca2+ immobilising agent TMB-8 does not affect binding of the signalling nucleotide, cAMP, to the cell surface receptors but abolishes the rapid accumulation of intracellular cGMP and subsequent chemotactic aggregation. We infer that movement of Ca2+ from membrane-bound stores is triggered by binding of cAMP to the cell-surface receptor and that this plays a primary role in stimulating cGMP formation and chemotaxis.     

The association of peroxisomes with the developing cell plate in dividing onion root cells depends on actin microfilaments and myosin

We have investigated changes in the distribution of peroxisomes through the cell cycle in onion (Allium cepa L.) root meristem cells with immunofluorescence and electron microscopy, and in leek (Allium porrum L.) epidermal cells with immunofluorescence and peroxisomal-targeted green fluorescent protein. During interphase and mitosis, peroxisomes distribute randomly throughout the cytoplasm, but beginning late in anaphase, they accumulate at the division plane. Initially, peroxisomes occur within the microtubule phragmoplast in two zones on either side of the developing cell plate. However, as the phragmoplast expands outwards to form an annulus, peroxisomes redistribute into a ring immediately inside the location of the microtubules. Peroxisome aggregation depends on actin microfilaments and myosin. Peroxisomes first accumulate in the division plane prior to the formation of the microtubule phragmoplast, and throughout cytokinesis, always co-localise with microfilaments. Microfilament-disrupting drugs (cytochalasin and latrunculin), and a putative inhibitor of myosin (2,3-butanedione monoxime), inhibit aggregation. We propose that aggregated peroxisomes function in the formation of the cell plate, either by regulating hydrogen peroxide production within the developing cell plate, or by their involvement in recycling of excess membranes from secretory vesicles via the -oxidation pathway. Differences in aggregation, a phenomenon which occurs in onion, some other monocots and to a lesser extent in tobacco BY-2 suspension cells, but which is not obvious in the roots of Arabidopsis thaliana (L.) Heynh., may reflect differences within the primary cell walls of these plants.Abbreviations BDM 2,3-butanedione monoxime - DAPI 4,6-diamidino-2-phenylindole - ER endoplasmic reticulum - GFP green fluorescent protein     

Downregulation of cell-to-cell communication by the viralsrc gene is blocked by TMB-8 and recovery of communication is blocked by vanadate

Summary The viralsrc gene downregulates junctional communication, closing cell-to-cell membrane channels presumably by way of the phosphoinositide signal route. We show that TMB-8 [8-N, N-(diethylamino) octyl-3,4,5-trimethoxybenzoate] counteracts this downregulation in cells transformed by temperature-sensitive mutant Rous sarcoma virus: TMB-8 (36–72 m) raises junctional permeability when applied during activity ofsrc protein kinase, i.e., at steady permissive temperature; and TMB-8 inhibits the fall of junctional permeability, when the activity ofsrc protein kinase gets turned on. TMB-8 also (reversibly) inhibits the growth of the cells at permissive temperature and reverses the morphological changes associated with transformation. The morphological reversal lags several hours behind the junctional-permeability reversal. Communication recovers within a few minutes when the activity of thesrc protein kinase is turned off (in absence of TMB-8). Sodium orthovanadate (20 m) prevents this recovery, but it has no major effect on junctional permeability on its own. We discuss possible modes of action of these agents on critical stages of the signal route, related to intracellular Ca²⁺ and protein kinase C.     

内钙拮抗剂TMB-8对白皮松花粉管细胞壁构建的影响

应用荧光显微技术、激光共聚焦扫描显微技术、单克隆抗体免疫荧光标记技术以及傅里叶变换显微红外光谱分析(FTIR)等手段,研究了内钙拮抗剂TMB-8对白皮松花粉管胞内Ca2+分布、花粉管生长以及细胞肇构建等的影响.结果表明,白皮松花粉管经TMB-8处理后,胞内的Ca2+浓度下降,花粉管内典型的Ca2+浓度梯度消失,花粉萌发...     

Membrane recycling occurs during asymmetric tip growth and cell plate formation inFucus distichus zygotes

Summary Zygotes of the brown algaFucus distichus undergo a series of intracellular changes resulting in the establishment of a polar growth axis prior to the first embryonic cell division. In order to examine the dynamics of membrane recycling which occur in the zygote during polar growth of the rhizoid, we probed living Fucus zygotes with the vital stain FM4-64, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylammo)phenyl)hexatrienyl)pyridinium dibromide. In newly fertilized, spherical zygotes, FM4-64 staining is symmetric and predominantly in the perinuclear region which is rich in endoplasmic reticulum, Golgi, and vacuolar membranes. As rhizoid or tip growth is initiated, this population of stained membranes becomes asymmetrically redistributed, concentrating at the rhizoid tip and extending centrally to the perinuclear region. This asymmetric localization is maintained in the zygote throughout polar growth of the rhizoid and during karyokinesis. Subsequently, FM4-64 staining also begins to accumulate in a central location between the daughter nuclei. As cytokinesis proceeds, this region of stain expands laterally from this central location, perpendicular to the plane of polar rhizoid outgrowth. The staining pattern thus delineates the formation of a cell plate, similar spatially to the accumulation of nascent plate membranes of higher plants. Treatment of Fucus zygotes with brefeldin-A inhibits both asymmetric growth of the rhizoid and formation of a new cell plate. These data suggest that inF. distichus FM4-64 is labeling a Golgi-derived membrane fraction that appears to be recycling between the site of tip growth, perinuclear region, and new cell plate.Abbreviations AF after fertilization - ASW artificial seawater - BFA brefeldin A - ER endoplasmic reticulum - FM4-64 N-(3-triethylam-moniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide     

Growth,cell wall formation and differentiation in the protonema of the moss,Funaria hygrometrica: Effects of plasmolysis on the developmental program and its expression

Summary Cultivation ofFunaria protonemata under plasmolytic or slightly subplasmolytic conditions initially causes a cessation of growth which is accompanied by a transient disappearance (or strong reduction in frequency, respectively) of putative cellulose synthesizing particle rosettes in the plasma membrane. Simultaneously, the formation and exocytosis of cell wall materialsecreting Golgi vesicles is slowed down. The latter process does not become apparent for several hours, though the reduction in activity can be proved indirectly. As a consequence of the imbalance between exocytosis, cell wall material accumulates in the plasmolytic space, generally at the cell tip. This indicates that the pattern of local, polar deposition of cell wall formation and cell elongation, membrane debris as well as wall material is maintained for some time. Later, however, the whole protoplast may become covered by new wall layers. Potentially growing filament tips and the distal region of nontip cells increase in diameter after longer cultivation in subplasmolytic conditions. It is suggested that normal wall growth results from a softening of the existing wall, its stretching and simultaneous stabilization by the apposition of new wall layers. We believe that the swelling is caused by a change in the equilibrium between the obviously less affected softening process and the imperfect stabilization by new wall layers because the wall layers which are formed at reduced turgor pressure are looser than normal and may have a changed composition.Kinetin-induced buds do not develop under plasmolytic conditions. Instead, spiral filaments are formed which readily give rise to buds when the osmotic value of the (kinetin-containing) medium is normalized. The results show that plasmolysis affects the expression of the developmental program rather than its initiation or maintenance.     

Effect of the intracellular Ca+2 antagonist TMB-8 on serum-stimulated Na+ influx in human fibroblasts

The effects of the intracellular Ca⁺² antagonist TMB-8 on the amiloride-sensitive Na⁺ influx pathway in human fibroblasts was investigated. It was found that TMB-8 inhibits serum- or growth factor-stimulated Na⁺ influx in a dose dependent fashion with a Ki value = 15 μM. A23187-stimulated Na⁺ influx on the other hand, was not inhibited by TMB-8. Furthermore, serum-stimulated Na⁺ influx could also be blocked by the calmodulin antagonist W-13. These results suggest that serum- or growth factor-stimulated Na⁺ influx is associated with an elevation of cytosolic free Ca⁺² levels, which then combine with calmodulin to activate the amiloride-sensitive Na⁺ influx pathway.     

Side branch formation and orientation in the caulonema of the moss,Funaria hygrometrica: Experiments with inhibitors and with centrifugation

Summary Colchicine treatment ofFunaria caulonemata, usually does not inhibit initiation of a side branch or its incipient elongation but does prevent movement of chloroplasts and the nucleus into the outgrowth. After colchicine and after cytochalasin B treatment side branches are formed about at the normal age of the cells; because of the inhibition of the apical cell they arise at an abnormal position,i.e., not in the third but in the second cell of a filament. After D₂O treatment the organelles are dislocated toward the basal cross wall. The site of side branch formation is then obviously determined by the position of the nucleus. Cells with an irreversibly reversed longitudinal polar axis can be found; by centrifugation in proximal direction the sites of side branch initiation likewise are displaced into the proximal region of the cell, especially if the remigration of the nucleus is inhibited by colchicine. High concentrations of Ca²⁺ ions induce the formation of side branch cells, without any outgrowth. The calcium ionophore A 23 187 influences the position of the nucleus and of the side branch only slightly. After these various treatments intercalary divisions frequently occur. The role and interrelationship of the nucleus and peripheral cytoplasm in establishing and maintaining the polar axes, and the role of microtubules are discussed.     

Endoplasmic reticulum in the formation of the cell plate and plasmodesmata

Summary The association of endoplasmic reticulum (ER) with the developing cell plate has been analyzed in lettuce roots fixed in glutaraldehyde and post-fixed in a mixture of osmium tetroxide-potassium ferricyanide (OsFeCN). Electron microscopic observations show that elements of ER, which are selectively stained by the OsFeCN reagent, become loosely associated with aggregating dictyosome vesicles at the onset of plate formation. Subsequently the ER, in a tubular reticulate network, surrounds the vesicular aggregates creating a three dimensional membrane matrix. It is suggested that the ER (1) provides a structural framework that holds the vesicles in position and directs their fusion within the plane of the plate and/or (2) regulates the local release of calcium ions required for vesicle fusion.OsFeCN post-fixation also provides new information about the cell plate vesicles themselves. The results demonstrate that vesicles derived from dictyosomes undergo an abrupt increase in staining as they fuse at the plate.Finally the ER associated with developing and mature plasmodesmata has been examined. Electron micrographs reveal that the OsFeCN staining, seen traversing the cell plate in early stages, later becomes restricted from that portion of the ER extending through the plasmodesmatal canal. These structural observations support the idea that during formation of the plasmodesma a tubular element of ER is tightly furled upon itself and that its inner leaflet is compressed into a rod. The ER cisternal space appears occluded and thus it is argued that intercellular transport occurs through the cytoplasmic annulus of the plasmodesmata.     

Production of 8-epi-tomentosin by plant cell culture ofXanthium strumarium

This study was conducted to establish a plant cell culture system for the production of medically important secondary metabolites fromXanthium strumarium. The effects of plant growth regulators including NAA, 2,4-D, kinetin, and ABA were examined in terms of callus induction, maintenance of callus and suspension cultures. It was shown that callus was induced upon treatment with NAA while embryo was induced after treatment with 2,4-D. Callus formation was further improved by treatment with ABA and NAA. The level of callusing increased by 17–29% for the seed case, cotyledon, leaf, and hypocotyl and by 96% in the case of the root. Suspension cell lines were established using calli produced from cotyledon, hypocotyl and root and cultured at 25°C under light conditions. The cells grew up to 15 g/L with NAA 2 ppm, BA 2 ppm, and ABA 1 ppm treatment. Supernatants of suspension cultures of cell lines derived from coyledon and hypocotyl produced some distinctive secondary metabolites, one of which was identified as 8-epi-tomentosin, which belongs to the xanthanolides. The amounts of 8-epi-tomentosin produced by the cotyledon-and hypocotylderived cell lines were 13.4 mg/L and 11.0 mg/L, respectively.     

Mechanism of plasmodesmata formation in characean algae in relation to evolution of intercellular communication in higher plants

It is generally accepted that higher plants evolved from ancestral forms of the modern charophytes. For this reason, we chose the characean alga, Chara corallina Klein ex Willd., em. R.D.W. (C. australis R. Br.), to determine whether this transition species produces plasmodesmata in a manner analogous to higher plants. As with higher plants and unlike most green algae, Chara utilizes a phragmoplast for cell division; however, in contrast with the situation in both lower and higher vascular plants, the developing cell plate and newly formed cell wall were found to be completely free of plasmodesmata. Only when the daughter cells had separated completely were plasmodesmata formed across the division wall. Presumably, highly localized activity of wall-degrading (or loosening) enzymes inserted into the plasma membrane play a central role in this process. In general appearance characean plasmodesmata are similar to those of higher plants with the notable exception that they lack an appressed endoplasmic reticulum. Further secondary modifications in plasmodesmal structure were found to occur as a function of cell development, giving rise to highly branched plasmodesmata in mature cell walls. These findings are discussed in terms of the evolution of the mechanism for plasmodesmata formation in algae and higher plants.This work was supported in part by National Foundation grant No. DCB-9016756 (W.J.L.). We thank the Electron Microscopy Center of Washington State University and the Zoology Department, University of California, Davis, for the use of their microscopy facilities.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> dynamin-related protein DRP2B is co-localized with DRP1A on the leading edge of the forming cell plate

The Arabidopsis genome has six families of dynamin-related proteins. One of these families includes DRP2A and DRP2B. The domain structures of proteins of this family are most similar to those of the animal endocytosis protein, dynamin. In this study, the signals of GFP-tagged DRP2B were strongly detected in the cell plate of Arabidopsis root tip cells and tobacco cultured cells. Time-lapse observations of these signals during cytokinesis in tobacco cultured cells suggested that DRP2B mainly localized to the newly formed part of the cell plate, and that the localization dynamics of DRP2B was quite similar to that of DRP1A, which is an Arabidopsis dynamin-related protein that is closely related to soybean phragmoplastin. These results indicate that Arabidopsis dynamin-related proteins, DRP1A and DRP2B, from two different families, participate in membrane remodeling at a similar place in the cell plate.     

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.     

Demarcation of the cortical division zone in dividing plant cells

Somatic cytokinesis in higher plants involves, besides the actual construction of a new cell wall, also the determination of a division zone. Several proteins have been shown to play a part in the mechanism that somatic plant cells use to control the positioning of the new cell wall. Plant cells determine the division zone at an early stage of cell division and use a transient microtubular structure, the preprophase band (PPB), during this process. The PPB is formed at the division zone, leaving behind a mark that during cytokinesis is utilized by the phragmoplast to guide the expanding cell plate toward the correct cortical insertion site. This review discusses old and new observations with regard to mechanisms implicated in the orientation of cell division and determination of a cortical division zone.     

The synthesis and biological properties of 8-azido-N 6-benzyladenine,a potential photoaffinity reagent for cytokinin

A cytokinin photoaffinity reagent, 8-azido-N ⁶-benzyladenine (8N₃BA), was synthesized from 8-bromoadenosine via azide replacement, benzylation at N–1, rearrangement to the N-6-benzyl derivative and acid hydrolysis. The compound thus obtained was found to have full cytokinin activity in the moss and tobacco cell-suspension bioassays. Photolysis of 8N₃BA was accomplished with long and short-wavelength ultraviolet light and produced compounds which had very little or no biological activity in the two bioassays. In-vivo photolysis of 8N₃BA caused loss of the cytokinin activity of this compound in moss protonemata. This result was similar to earlier ones where the biological response of moss protonemata to benzyladenine was reversed following removal of the hormone by a short rinse with water.Abbreviations BA N ⁶-benzyladenine - 8N₃BA 8-azido-N ⁶-benzyladenine - PMR proton magnetic resonance - TLC thin-layer chromatography - UV ultraviolet In partial fulfillment of requirements for the Ph.D. degree at Michigan State University     

BAPTA-calcium buffers modulate cell plate formation in stamen hairs ofTradescantia: evidence for calcium gradients

Summary Five BAPTA buffers with differential affinities for Ca²⁺ have been examined for their effects on cell plate formation in stamen hair cells ofTradescantia. The five include 5,5-dimethyl BAPTA (Kd=0.15 M), BAPTA (Kd=0.22 M), 5,5-dibromo BAPTA (Kd=1.5 M), 5-methyl,5-nitro BAPTA (Kd=22 M), and 5-nitro BAPTA (Kd=40 M). At a concentration of 5 mM and 25 mM in the pipette, the buffers were iontophoretically microinjected into dividing stamen hair cells (2 nA for 1 min) prior to or at the onset of cell plate formation. At the lowest concentration (5 mM), only one buffer, 5,5-dibromo BAPTA, inhibits cell plate formation, and is most effective if delivered at the moment of cell plate vesicle aggregation. The inhibitory effects appear as a slowing of cell plate expansion, the formation of distorted plates, or the complete dissolution of plates that might have initiated normally. When the pipette tip concentration is elevated to 25 mM, the effects of 5,5-dibromo BAPTA become more profound. At these levels 5,5-dimethyl BAPTA, BAPTA, and 5-nitro BAPTA also modulate cell plate formation, producing effects similar to that of 5,5-dibromo BAPTA at the lower concentration. Independent studies using fura-2 as a fluorescent analogue of the BAPTA buffers, indicate that the apparent effective concentration for 5,5-dibromo BAPTA is between 1.0–1.4 mM; its threshold concentration is not known but expected to be somewhat lower. For the other buffers the threshold concentration is between 1.5–2.2 mM. The concentration dependence supports the idea that the buffers facilitate diffusion of Ca²⁺ away from regions of elevated concentration. The results thus provide evidence that local Ca²⁺ gradients may be present in the vicinity of the cell plate and that they participate in the cytokinetic process.Dedicated to the memory of Professor John G. Torrey     

Expression of the JIM8 cell wall epitope in carrot somatic embryogenesis

Certain single cells in carrot (Daucus carota L.) suspension cultures react with the monoclonal antibody JIM8, and it has been proposed that these cells represent a transitional stage in somatic embryo formation. Shortly after isolation of the single cells by sieving, up to 80% of the cells react with JIM8. Within 4 d, JIM8 labelling becomes restricted to 1% of the single cells. To obtain evidence for the proposed correlation between expression of the JIM8 cell wall epitope and somatic embryo formation the developmental fate of carrot single cells labelled with JIM8 was determined by cell tracking. The results, obtained by recording 43 000 cells, show that only few JIM8-labelled cells give rise to embryos, and most somatic embryos develop from cells devoid of the JIM8 cell wall epitope. We therefore conclude that the presence of the JIM8 cell wall epitope does not coincide with the ability of single suspension cells to form embryos.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - AGP arabino galactan protein - B5-0 Gamborg's B5 medium - B5-0.2 Gamborg's B5 medium supplemented with 0.2 M 2,4-D - FITC fluoresceïn isothiocyanate - PBS phosphate-buffered saline     

The herbicide dichlobenil disrupts cell plate formation: immunogold characterization

Summary We have utilized light and transmission electron microscopy and immunocytochemistry to examine onion roots treated with the herbicide dichlobenil (2,6-dichlorobenzonitrile; DCB), a purported disrupter of cellulose biosynthesis. The most salient effect of DCB is observed on cell plate formation, the process that gives rise to new cell walls. In the presence of DCB, cell plates develop normally up to the tubular network stage. They are the result of fusion of Golgi-derived vesicles and the accumulation of callose and the first strands of cellulose. The DCB-treated cell plates retain the reticulate and malleable nature of the tubular network/early fenestrated plate stage of cell plate formation, but fail to display signs of the stiffening and straightening associated with an accumulation of cellulose. Instead, the malleable cell plates in the DCB-treated cells retain a wavy architecture, accumulate pockets of electron opaque material, and produce plasmodesmata in abnormal orientations. Immunocytochemical investigations of the abnormal cell plates formed after DCB treatment show 20-fold increase in the level of callose labelling found in the control cell plates. Xyloglucans and rhamnogalacturonans can be detected in the partially-formed cell plates, with the labelling density of xyloglucan 4–5 times greater than in the control cell plates and that of the rhamnogalacturonans being similar to the controls. These data support the hypothesis that DCB inhibits cellulose biosynthesis as a primary mechanism of action, and that in the absence of cellulose synthesis the cell plates fail to mature and to give rise to new cross walls.Abbreviations DCB dichlorobenzonitrile - PGA/RGI polygalacturonic acid/rhamnogalacturonan I     

Side branch formation and orientation in the caulonema of the moss,Funaria hygrometrica: Normal development and fine structure

Summary The regular branching of theFunaria caulonema filaments is partly related to rhythms in nuclear and cell division. The formation and development of the branches were studied by light and electron microscopy with particular attention directed to the distribution of microtubules and the polar organization of the cytoplasm. The new side branch breaks through the wall of the mother cell. The site of branch development is determined by the position of the nucleus of the mother cell. In protonemata which grow in vertically placed Petri dishes gravity influences the position of nuclei and side branches, and also the direction of oblique cross walls in the caulonema filaments to a certain extent.