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.     

Formation of a phragmosome-like structure in centrifuged protonemal cells of Adiantum capillus-veneris L.

A phragmosome (PS) is a transvacuolar aggregation of cytoplasm that develops in the plane of future cytokinesis and is found specifically in highly vacuolated cells. Although protonemal cells of Adiantum capillus-veneris L. usually do not form a PS, a PS-like structure developed at the site of a preprophase band (PPB) of microtubules (MTs) when the nucleus and endoplasm were displaced from the division site by centrifugation, leaving a PPB in the cortical cytoplasm. The PS-like structure contained endoplasmic MTs, F-actin, oil droplets and mitochondria. The structure did not develop when the cells were centrifuged before the formation of a PPB. Application of amiprophos-methyl (APM) before development of the PPB strongly inhibited the formation of the PS-like structure after centrifugation. The PS-like structure was dispersed after cytokinesis which occurred in the region of the displaced nucleus. Treatment with APM after the formation of the PS-like structure arrested the cell cycle at the M phase and inhibited the degradation of this structure. These results suggest that development of a PS-like structure is associated both with the formation of a PPB and with the stage of the cell cycle. Received: 9 July 1996 / Accepted: 12 September 1996     

EFFECT OF CENTRIFUGATION ON THE DEVELOPMENT AND TIMING OF PREMITOTIC POSITIONING OF THE NUCLEUS IN ADIANTUM PROTONEMATA

When single-celled protonemata of Adiantum capillus-veneris L. were centrifuged immediately before transferring to darkness from continuous irradiation with red light, their nuclei were displaced basipetally. Both filamentous and branched protonemata were obtained. The stronger the centrifugal acceleration, the more frequently the branched protonemata were induced.
The effect of centrifugation at 1,300 x g for 15 min on nuclear displacement was different at different stages of the cell cycle. In early G₁ phase, the nucleus was easily displaced by centrifugation, but quickly returned to the original position after centrifugation. In late G₁ phase, the nucleus was displaced, but after centrifugation it never came back to the original position. In late G₂ and M phases, the nucleus was no longer displaced by the centrifugation. Premitotic positioning of the nucleus in cytokinesis took place about 5 hr before cell plate formation in all centrifugal treatments described above.     

Presence and absence of the preprophase band of microtubules in moss protonemata: a clue to understanding its function?

Summary InFunaria protonemata, preprophase bands (PPBs) of microtubules do not develop when the tip cell divides, when side branches are initiated or in intercalary regeneration divisions. We report here that PPBs do, however, develop when a tmema cell is formed. In the former cases, cell division is not coupled with an expansion of the mother cell wall at the site where the cell plate will attach. In the latter case, the mother cell wall ruptures at that site and the tmema cell elongates. This observation and the findings on presence and absence of the PPB in other cell types indicate a connection between PPB occurrence and mother cell wall expansion. They support the idea that the PPB might be involved in the local secretion of cell wall material. We extend this notion, suggesting that the microtubules of the PPB control the oriented deposition of a thin layer of cellulose microfibrils at the mother cell wall which supports the firm attachment of the cell plate when the mother cell wall expands.Abbreviations FITC fluorescein isothiocyanate - IgG immunoglobulin G - MT microtubule - PPB preprophase band of microtubules - TC tmema cell     

Intracellular Photoreceptive Site for Blue Light-Induced Cell Division in Protonemata of the Fern Adiantum--Further Analyses by Polarized Light Irradiation and Cell Centrifugation

The intracellular localization of the photoreceptive site forblue light-induced cell division in single-celled protonemataof Adiantum capillus-veneris L. was investigated using polarizedlight irradiation and protonemal cell centrifugation. The responseto irradiation with polarized blue light showed no dependenceon the direction of light polarization. However, centrifugationof the protonemata followed by microbeam irradiation showedthat the site of blue light perception could be displaced togetherwith the nucleus. Centrifugal treatment changed the distributionof intracellular organelles at the time of light exposure andbasipetally displaced the nucleus about 90µm. This treatmenthad no effect on the induction of cell division with blue lightif the protonemata were centrifuged again acropetally afterthe light treatment. Microbeam (30x30 µm²) irradiationwith blue light of the apical 45–75 ßm region,the receptive site of blue light in non-centrifuged cell, didnot induce cell division. However, cell division was inducedby irradiation of the nucleus-containing region, indicatingthat the photoreceptive site was displaced together with thenucleus by the centrifugation. These results suggest that theblue light receptor regulating cell division in Adiantum protonematais not likely to be located on the plasma membrane. (Received February 20, 1986; Accepted May 27, 1986)     

Decision of spindle poles and division plane by double preprophase bands in a BY-2 cell line expressing GFP-tubulin

The preprophase band (PPB) of microtubules is thought to be involved in deciding the future division site. In this study, we investigated the effects of double PPBs on spindle formation and the directional decision of cytokinesis by using transgenic BY-2 cells expressing green fluorescent protein (GFP)-tubulin. At prophase, most of the cells with double PPBs formed multipolar spindles, whereas all cells with single PPBs formed normal bipolar spindles, clearly implicating the PPB in deciding the spindle poles. At metaphase, however, both cell types possessed the bipolar spindles, indicating the existence of correctional mechanism(s) at prometaphase. From prometaphase to metaphase, the spindles in double PPB cells altered their directions to become oblique to the cell-elongating axis, and these orientations were maintained in the phragmoplast and resulted in the oblique division planes. These oblique cell plates decreased when actin microfilaments were disrupted, and double actin-depleted zones (ADZs) appeared where the double PPBs had existed. These results suggest that the information necessary for proper cytokinesis may be transferred from the PPBs to the ADZs, even in the case of the double PPBs.     

Re-Formation of the Preprophase Band After Cold-Induced Depolymerization of Microtubules in Adiantum Protonemata

Re-formation of the preprophase band (PPB) of microtubules (MTs)after cold-induced depolymerization of MTs was investigatedin protonemal cells of the fern, Adiantum capiilus-veneris L.When protonemata with a PPB were chilled at 0?C, MTs of thePPB depolymerized within 5 min leaving only a few fragmentsof MTs, and all such fragments were disrupted within 2 h afterthe onset of cold treatment. When cells were chilled for 5 minand then rewarmed at 25 ?C, the transverse MTs of the PPB graduallyincreased in number in the region of the PPB and re-formationof the PPB was accomplished within 20 min. In contrast, whencells chilled for 2 h were rewarmed, randomly oriented MTs appearedinitially and then a PPB with a low density of MTs became apparent.The PPB re-formed even when the nucleus and most of the endoplasmhad been displaced from the region of the PPB by centrifugation(2,800 ?g, 15 min). These results suggest that MTs of the PPBnucleate and are organized in the cortical cytoplasm duringre-formation of the PPB after disruption. (Received June 27, 1991; Accepted August 15, 1991)     

Preprophase bands in cultured multinucleate soybean protoplasts

Summary Multinucleate cells derived from soybean protoplasts were used to investigate the effect of increased nuclear number on the development and frequency of preprophase bands (PPBs) of microtubules (MTs). The results do not support the assumption that one nucleus establishes one PPB because the majority of multinucleate cells had only one large PPB. However, nuclear number or ploidy level has some influence on PPB development since double PPBs occurred more often in multinucleate than uninucleate cells. Double (divergent) PPBs were present at early and late stages of PPB development, suggesting that they are not a transient stage. PPBs in multinucleate cells developed in a similar fashion to those in uninucleate cells. In multinucleate cells, each dividing nucleus had its own spindle and phragmoplast. Subsequent phragmoplast development was frequently uncoupled from PPB distribution. Most multinucleates contained a single large PPB but at telophase, multiple phragmoplasts oriented in different planes.Abbreviations MT microtubule - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline - PNF perinuclear fluorescence - PPB preprophase band     

Rearrangements of Microtubules Involved in Establishing Cell Division Planes Start Immediately after DNA Synthesis and Are Completed just before Mitosis

This work concerns an aspect of spatial regulation of cell division, the development of the preprophase band (PPB) of microtubules. The PPB is significant in plant development because its position in the dividing cell indicates where the new cell wall will be inserted[mdash]an important site for control of histogenesis. We have categorized and determined the durations of stages in the development of PPBs, and have established their timing relative to the S-, G2-, and mitotic phases of the cell cycle. Roots of wheat seedlings were supplied with bromodeoxyuridine in continuous and pulse-chase treatments. Cells that were in the S-phase were identified and changes in their microtubule arrays were monitored by double immunolabeling. PPB initiation was detectable as early as the end of the S-phase as a narrowing of the preceding interphase array of microtubules. Development continued throughout G2 to a mature, narrow PPB, which existed only briefly and then eroded during the transition to the prophase mitotic spindle. The microtubule rearrangements of PPB development showed that preparation of the future site and plane of division in higher plant cells begins just after DNA replication and is completed just before mitosis.     

Actomyosin is Involved in the Organization of the Microtubule Preprophase Band in Arabidopsis Suspension Cultured Cells

The microtubule preprophase bands （PPBs） participate in the sequence of events to position cell plates in most plants. However, the mechanism of PPB formation remains to be clarified. In the present study, the organization of PPBs in Arabidopsis suspension cultured cells was investigated by confocal laser scanning microscopy combined with pharmacological treatments of reagents specific for the cytoskeleton elements. Double staining of F-actin and microtubules （MTs） showed that actin filaments were arranged randomly and no colocalization with cortical MTs was observed in the interphase cells. However, cortical actin filaments showed colocalization with MTs during the formation of PPBs. A broad actin band formed with the broad MT band in the initiation of PPB and narrowed down together with the MT band to form the PPB. Nevertheless, broad MT bands were formed but failed to narrow down in cells treated with the F-actin disruptor latrunculin A. In contrast, in the presence of the F-actin stabilizer phalloidin, PPB formation did not exhibit any abnormality. Therefore, the integrity, but not the dynamics, of the actin cytoskeleton is necessary for the formation of normal PPBs. Treatment with 2, 3-butanedine monoxime, a myosin inhibitor, also resulted in the formation of broad MT bands, indicating that actomyosin may be involved in the rearrangement of MTs to form the PPBs. Double staining of MTs and myosin revealed that myosin concentrated on the PPB region during PPB formation. It is suggested that the actin cytoskeleton at the PPB site may serve as a rack to transport cortical MTs by using myosin when the broad MT band narrows down to form the PPB.     

Development of the preprophase band from random cytoplasmic microtubules in guard mother cells of Allium cepa L.

The organization of microtubule (MT) arrays in the guard mother cells (GMCs) of A. cepa was examined, focussing on the stage at which a longitudinal preprophase band (PPB) is established perpendicular to all other division planes in the epidermis. In the majority of young GMCs, including those seen just after asymmetric division, MTs are distributed randomly throughout the cortex and inner regions of the cytoplasm. Few MTs are associated with the nuclear surface. As the GMCs continue to develop, MTs cluster around the nucleus and a PPB appears as a wide longitudinal band. Microtubules also become prominent between the nucleus and the periclinal and transverse walls, while they decrease in number along the radial longitudinal walls. The PPB progressively narrows by early prophase, and a transversely oriented spindle gradually ensheaths the nucleus. These observations indicate that the initial, broad PPB is organized by a rearrangement of the random cytoplasmic array of MTs. Additional reorganization is responsible for MTs linking the nucleus and the cortex in the future plane of the cell plate, and for narrowing of the PPB.Abbreviations GMC guard mother cell - MT microtubule - PPB preprophase band     

Nuclear behavior during branch formation in a centrifugedAdiantum protonema and the nuclear polarity

A new branch was induced on the side wall of fern protonema by cell centrifugation and subsequent polarized red light irradiation after the induction of cell division under white light. Nuclear behavior during the branch formation was analyzed. Immediately after cell division, the two daughter nuclei moved away from the division site in both red and dark conditions. Under continuous irradiation with polarized red light, cell swelling occurred as an early step of branching near the cell dividing wall, even though the nucleus was localized far from the branching site at the beginning of the swelling. After a new branch started to grow, the nucleus returned to the branching site and moved into the new branch from its basipetal end. When a protonema incubated in the dark was centrifuged again acropetally or basipetally just before the irradiation of polarized red light, the rate of apical growth or branch formation was increased, respectively. Moreover, growth of a branched protonema was altered from its former apex or from the branch again by dislocating the nucleus acropetally or basipetally by centrifugation, respectively. These facts suggest that the nucleus has no polarity physiologically, i.e. head and tail, namely either end of the spindle-shaped nucleus can be the nuclear front in a tip-growing protonema.     

Microtubule arrays and Arabidopsis stomatal development

Microtubule arrays in living cells were analysed during Arabidopsis stomatal development in order to more closely define stages in the pathway and contexts where intercellular signalling might operate. Arabidopsis stomata are patterned iteratively via the orientation of an asymmetric division in a cell located next to an existing stoma. It was found that preprophase bands of microtubules (PPBs) were correctly placed away from stomata and from two types of precursor cells. This suggests that all three cell types participate in an intercellular signalling pathway that orients the division site. These and other asymmetric divisions in the pathway were preceded by a polarized cytoplasm, with the PPB around the nucleus at one end, and the vacuole at the other. PPBs before symmetric divisions of guard mother cells (GMCs) were broader than those in asymmetric divisions, and the GMC division site was marked by unusual end-wall thickenings. This work identifies an accessible system for studying cytoskeletal function and provides a foundation for analysing the role of genes involved in stomatal development.     

Preprophase bands of microtubules and the cell cycle: Kinetics and experimental uncoupling of their formation from the nuclear cycle in onion root-tip cells

We have studied the timing of preprophase band (PPB) development in the division cycle of onion (Allium cepa L.) root-tip cells by combinations of immunofluorescence microscopy of microtubules, microspectrophotometry of nuclear DNA, and autoradiography of [³H]thymidine incorporation during pulse-chase experiments. In normally grown onion root tips, every cell with a PPB had the G2 level of nuclear DNA. Some were in interphase, prior to chromatin condensation, and some had varying degrees of chromatin condensation, up to the stage of prophase at which the PPB-prophase spindle transition occurs. In addition, autoradiography showed that PPBs can be formed in cells which have just finished their S phase, and microspectrophotometry enabled us to detect a population of cells in G2 which had no PPBs, these presumably including cells which had left the division cycle. The effects of inhibitors of DNA synthesis showed that the formation of PPBs is not fully coupled to events of the nuclear cycle. Although the mitotic index decreased 6-10-fold to less than 0.5% when roots were kept in 20 g·ml^-1 aphidicolin for more than 8 h, the percentage of cells containing PPBs did not decrease in proportion: the number of cells in interphase with PPBs increased while the number in prophase decreased. Almost the same phenomena were observed in the presence of 100 g·ml^-1 5-aminouracil and 40 g·ml^-1 hydroxyurea. In controls, all cells with PPBs were in G2 or prophase, but in the presence of aphidicolin, 5-aminouracil or hydroxyurea, some of the interphase cells with PPBs were in the S phase or even in the G1 phase. We conclude that PPB formation normally occurs in G2 (in at least some cases very early in G2) and that this timing can be experimentally uncoupled from the timing of DNA duplication in the cell-division cycle. The result accords with other evidence indicating that the cytoplasmic events of cytokinesis are controlled in parallel to the nuclear cycle, rather than in an obligatorily coupled sequence.Abbreviations APC aphidicolin - 5-AU 5-aminouracil - DAPI 4, 6-diamidino-2phenylindole - HU hydroxyurea - MI mitotic index - MT microtubule - PMSF phenylmethyl-sulfonyl fluoride - PPB preprophase band - %PPB percentage of cells with PPBs     

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.     

Arabidopsis TANGLED identifies the division plane throughout mitosis and cytokinesis

BACKGROUND: In premitotic plant cells, the future division plane is predicted by a cortical ring of microtubules and F-actin called the preprophase band (PPB). The PPB persists throughout prophase, but is disassembled upon nuclear-envelope breakdown as the mitotic spindle forms. Following nuclear division, a cytokinetic phragmoplast forms between the daughter nuclei and expands laterally to attach the new cell wall at the former PPB site. A variety of observations suggest that expanding phragmoplasts are actively guided to the former PPB site, but little is known about how plant cells "remember" this site after PPB disassembly. RESULTS: In premitotic plant cells, Arabidopsis TANGLED fused to YFP (AtTAN::YFP) colocalizes at the future division plane with PPBs. Strikingly, cortical AtTAN::YFP rings persist after PPB disassembly, marking the division plane throughout mitosis and cytokinesis. The AtTAN::YFP ring is relatively broad during preprophase/prophase and mitosis; narrows to become a sharper, more punctate ring during cytokinesis; and then rapidly disassembles upon completion of cytokinesis. The initial recruitment of AtTAN::YFP to the division plane requires microtubules and the kinesins POK1 and POK2, but subsequent maintenance of AtTAN::YFP rings appears to be microtubule independent. Consistent with the localization data, analysis of Arabidopsis tan mutants shows that AtTAN plays a role in guidance of expanding phragmoplasts to the former PPB site. CONCLUSIONS: AtTAN is implicated as a component of a cortical guidance cue that remains behind when the PPB is disassembled and directs the expanding phragmoplast to the former PPB site during cytokinesis.     

Preprophase bands in a suspension culture of the monocot Spartina pectinata

Continuous suspension cultures of the marsh grass Spartina pectinata grow as either unorganized colonies or files of cells. Immunofluorescence of tubulin revealed microtubule (MT) structures similar to those encountered in meristematic cells, including cortical microtubule (MT) bands in some interphase cells and in all prophase cells. These MT bands were judged to be preprophase bands (PPBs) on the basis of their temporal appearance in the cell cycle and their position and orientation relative to division planes. Although PPBs are widely thought to be associated with organized tissues and polarized divisions, there are reports of PPBs in suspension cultures of four dicot species. This is the first report of a PPB in suspension cultures of a monocot species.     

Is the preprophase band of microtubules a marker of organization in suspension cultures?

Summary To date it has been accepted that preprophase bands of microtubules (PPBs) either do not precede cell division or do so inconsistently in suspension cultures, the assumption being that such cultures proliferate in an unorganized state in which placement of cell plates is not regulated by the PPB system that is widespread in organized tissues. Using indirect immunofluorescence microscopy with antitubulin, the relative frequency of occurrence of PPBs in enzymatically separated cells from root tips and suspension cultures of carrot and tobacco, was quantified by taking the ratio of the number of PPBs: phragmoplast. This ratio was termed the PPB index.One carrot suspension culture proliferated in a medium containing 2,4-Dichlorophenoxyacetic acid (2,4-D), and recognizable stages in somatic embryogenesis formed when 2,4-D was removed from the medium. Another carrot suspension culture was nonembryogenic and removal of 2,4-D resulted in a reduction of cell division and increase in cell elongation. The tobacco culture was a cytokinin habituated cell line and also required 2,4-D to maintain cell division. It ceased proliferation, and cell elongation took place if 2,4-D was removed.The PPB index in the root tips from both species, and in both types of carrot suspension culture was approximately the same but was approx. 15-fold lower in the tobacco suspension. PPBs in the tobacco suspension were atypical in structure as well as sparse in numbers. The PPB index allows quantitative comparisons between different tissues to be made. The low PPB index and the irregular PPBs in the tobacco suspension correlates with its inability to undergo organized morphogenesis and generate spatially defined cell lineages upon 2,4-D removal. In contrast, the high PPB index in the carrot suspension cultures correlates with their potential for organized embryo formation, whether or not that potential is realized by withdrawal of 2,4-D. However, their high PPB index is not obligatorily coupled to embryogenesis.     

Phragmoplasts in the Absence of Nuclear Division

All land plants (embryophytes) use a phragmoplast for cytokinesis. Phragmoplasts are distinctive cytoskeletal structures that are instrumental in the deposition of new walls in both vegetative and reproductive phases of the life cycle. In meristems, the phragmoplast is initiated among remaining non-kinetochore spindle fibers between sister nuclei and expands to join parental walls at the site previously marked by the preprophase band of microtubules (PPB). The microtubule cycle and cell cycle are closely coordinated: the hoop-like cortical microtubules of interphase are replaced by the PPB just prior to prophase, the PPB disappears as the spindle forms, and the phragmoplast mediates cell plate deposition after nuclear division. In the reproductive phase, however, cortical microtubules and PPBs are absent and cytokinesis may be uncoupled from the cell cycle resulting in multinucleate cells (syncytia). Minisyncytia of 4 nuclei occur in microsporocytes and several (typically 8) nuclei occur in the developing megagametophyte. Macrosyncytia with thousands of nuclei may occur in the nuclear type endosperm. Cellularization of syncytia involves formation of adventitious phragmoplasts at boundaries of nuclear-cytoplasmic domains (NCDs) defined by radial microtubule systems (RMSs) emanating from non-sister nuclei. Once initiated in the region of microtubule overlap at interfaces of opposing RMSs, the adventitious phragmoplasts appear structurally identical to interzonal phragmoplasts. Phragmoplasts are constructed of multiple opposing arrays similar to what have been termed microtubule converging centers. The individual phragmoplast units are distinctive fusiform bundles of anti-parallel microtubules bisected by a dark mid-zone where vesicles accumulate and fuse into a cell plate.     

The effect of taxol onTriticum preprophase root cells: preprophase microtubule band organization seems to depend on new microtubule assembly

Summary To examine whether preprophase microtubule band (PPB) organization occurs by rearrangement of pre-existing, or by assembly of new microtubules (Mts), we treated root cells ofTriticum turgidum with taxol, which stabilizes pre-existing Mts by slowing their depolymerization. With taxol early preprophase cells failed to form a normal PPB and PPB narrowing was prevented in cells that had already formed a wide one. The PPB became persistent in prometaphase cells and the formation of multipolar prophase-prometaphase spindles was induced. These data favour the suggestion that PPB formation and narrowing, as well as prophase spindle development, are dynamic processes depending on continuous Mt assembly at the PPB site and in the perinuclear cytoplasm.Abbreviations Mt microtubule - MTOC microtubule organizing centre - PPB preprophase microtubule band - DMSO dimethyl sulfoxide