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Key message: Plants exhibit a unique pattern of cytosolic Ca2+ dynamics to correlate with microtubules to regulate cytokinesis, which significantly differs from those observed in animal and yeast cells. Calcium (Ca2+) transients mediated signaling is known to be essential in cytokinesis across eukaryotic cells. However, the detailed spatiotemporal dynamics of Ca2+ during plant cytokinesis remain largely unexplored. In this study, we employed GCaMP5, a genetically encoded Ca2+ sensor, to investigate cytokinetic Ca2+ transients during cytokinesis in Nicotiana tabacum Bright Yellow-2 (BY-2) cells. We validated the effectiveness of GCaMP5 to capture fluctuations in intracellular free Ca2+ in transgenic BY-2 cells. Our results reveal that Ca2+ dynamics during BY-2 cell cytokinesis are distinctly different from those observed in embryonic and yeast cells. It is characterized by an initial significant Ca2+ spike within the phragmoplast region. This spike is followed by a decrease in Ca2+ concentration at the onset of cytokinesis in phragmoplast, which then remains elevated in comparison to the cytosolic Ca2+ until the completion of cell plate formation. At the end of cytokinesis, Ca2+ becomes uniformly distributed in the cytosol. This pattern contrasts with the typical dual waves of Ca2+ spikes observed during cytokinesis in animal embryonic cells and fission yeasts. Furthermore, applications of pharmaceutical inhibitors for either Ca2+ or microtubules revealed a close correlation between Ca2+ transients and microtubule organization in the regulation of cytokinesis. Collectively, our findings highlight the unique dynamics and crucial role of Ca2+ transients during plant cell cytokinesis, and provides new insights into plant cell division mechanisms. [ABSTRACT FROM AUTHOR]

Main conclusions: STD1 specifically interacts with MAP65-5 in rice and they cooperatively control microtubule bundles in phragmoplast expansion during cell division. Microtubules play critical roles during the cell cycle progression in the plant cell. We previously reported that STEMLESS DWARF 1 (STD1), a kinesin-related protein, was localized specifically to the phragmoplast midzone during telophase to regulate the lateral expansion of phragmoplast in rice (Oryza sativa). However, how STD1 regulates microtubule organization remains unknown. Here, we found that STD1 interacted directly with MAP65-5, a member of the microtubule-associated proteins (MAPs). Both STD1 and MAP65-5 could form homodimers and bundle microtubules individually. Compared with MAP65-5, the microtubules bundled by STD1 were disassembled completely into single microtubules after adding ATP. Conversely, the interaction of STD1 with MAP65-5 enhanced the microtubule bundling. These results suggest STD1 and MAP65-5 might cooperatively regulate microtubule organization in the phragmoplast at telophase. [ABSTRACT FROM AUTHOR]

Advances in cell biology have been largely driven by pioneering work in model systems, the majority of which are from one major eukaryotic lineage, the opisthokonts. However, with the explosion of genomic information in many lineages, it has become clear that eukaryotes have incredible diversity in many cellular systems, including the cytoskeleton. By identifying model systems in diverse lineages, it may be possible to begin to understand the evolutionary origins of the eukaryotic cytoskeleton. Within the plant lineage, cell biological studies in the model moss, Physcomitrella patens, have over the past decade provided key insights into how the cytoskeleton drives cell and tissue morphology. Here, we review P. patens attributes that make it such a rich resource for cytoskeletal cell biological inquiry and highlight recent key findings with regard to intracellular transport, microtubule-actin interactions, and gene discovery that promises for many years to provide new cytoskeletal players. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are emerging players in several biological processes. The present work investigates their potential involvement in plant cytokinesis by the application of reagents disturbing ROS homeostasis in root-tip cells of Triticum turgidum. In particular, the NADPH-oxidase inhibitor diphenylene iodonium, the ROS scavenger N-acetyl-cysteine, and menadione that leads to ROS overproduction were used. The effects on cytokinetic cells were examined using light, fluorescence, and transmission electron microscopy. ROS imbalance had a great impact on the cytokinetic process including the following: (a) formation of atypical 'phragmoplasts' incapable of guiding vesicles to the equatorial plane, (b) inhibition of the dictyosomal and/or endosomal vesicle production that provides the developing cell plates with membranous and matrix polysaccharidic material, (c) disturbance of the fusion processes between vesicles arriving on the cell plate plane, (d) disruption of endocytic vesicle production that mediates the removal of the excess membrane material from the developing cell plate, and (e) the persistence of large callose depositions in treated cell plates. Consequently, either elevated or low ROS levels in cytokinetic root-tip cells resulted in a total inhibition of cell plate assembly or the formation of aberrant cell plates, depending on the stage of the affected cytokinetic cells. The latter failed to expand towards cell cortex and hence to give rise to complete daughter cell wall. These data revealed for the first time the necessity of ROS homeostasis for accomplishment of plant cytokinesis, since it seems to be a prerequisite for almost every aspect of this process. [ABSTRACT FROM AUTHOR]

During cytokinesis the cytoplasm of a cell is divided to form two daughter cells. In animal cells, the existing plasma membrane is first constricted and then abscised to generate two individual plasma membranes. Plant cells on the other hand divide by forming an interior dividing wall, the so-called cell plate, which is constructed by localized deposition of membrane and cell wall material. Construction starts in the centre of the cell at the locus of the mitotic spindle and continues radially towards the existing plasma membrane. Finally the membrane of the cell plate and plasma membrane fuse to form two individual plasma membranes. Two microtubule-based cytoskeletal networks, the phragmoplast and the pre-prophase band (PPB), jointly control cytokinesis in plants. The bipolar microtubule array of the phragmoplast regulates cell plate deposition towards a cortical position that is templated by the ring-shaped microtubule array of the PPB. In contrast to most animal cells, plants do not use centrosomes as foci of microtubule growth initiation. Instead, plant microtubule networks are striking examples of self-organizing systems that emerge from physically constrained interactions of dispersed microtubules. Here we will discuss how microtubule-based activities including growth, shrinkage, severing, sliding, nucleation and bundling interrelate to jointly generate the required ordered structures. Evidence mounts that adapter proteins sense the local geometry of microtubules to locally modulate the activity of proteins involved in microtubule growth regulation and severing. Many of the proteins and mechanisms involved have roles in other microtubule assemblies as well, bestowing broader relevance to insights gained from plants. [ABSTRACT FROM AUTHOR]

BS366 is a thermo-sensitive male sterile line of wheat ( Triticum aestivum L.) for two-line hybrid breeding, which exhibits aberrant meiotic cytokinesis under low temperature. Through transcriptome analysis, a possible regulatory role for plant actin cytoskeleton was suggested. However, the organization of actin cytoskeleton in meiosis has been poorly understood so far. Here, fixed microsporocytes during meiosis were labeled with tetramethylrhodamine isothiocyanate-phalloidin and 4′,6-diamidino-2-phenylindole. Quantities of fluorescent micrographs were captured using a confocal microscope, including the transient state from metaphase to telophase. We observed that actin filaments were abundant in typical kariokinetic spindle, central spindle (parallel microtubules or actin fibers between two separated chromosomes in anaphase), and phragmoplast. Interestingly, we identified the Chinese lantern-shaped actin phragmoplast in wheat meiosis for the first time. Under low temperature, phragmoplast actin filaments were chaotic and normal cell plate failed to form. These data provide new insights into the organization of actin filaments during male meiosis of plant and support a role of actin cytoskeleton in bringing about thermo-sensitive male sterility in wheat. [ABSTRACT FROM AUTHOR]

Plants possess a large number of microtubule-based kinesin motor proteins. While the kinesin-2, 3, 9, and 11 families are absent from land plants, the kinesin-7 and 14 families are greatly expanded. In addition, some kinesins are specifically present only in land plants. The distinctive inventory of plant kinesins suggests that kinesins have evolved to perform specialized functions in plants. Plants assemble unique microtubule arrays during their cell cycle, including the interphase cortical microtubule array, preprophase band, anastral spindle and phragmoplast. In this review, we explore the functions of plant kinesins from a microtubule array viewpoint, focusing mainly on Arabidopsis kinesins. We emphasize the conserved and novel functions of plant kinesins in the organization and function of the different microtubule arrays. [ABSTRACT FROM AUTHOR]

Coordinated cell divisions and cell expansion are the key processes that command growth in all organisms. The orientation of cell divisions and the direction of cell expansion are critical for normal development. Symmetric divisions contribute to proliferation and growth, while asymmetric divisions initiate pattern formation and differentiation. In plants these processes are of particular importance since their cells are encased in cellulosic walls that determine their shape and lock their position within tissues and organs. Several recent studies have analyzed the relationship between cell shape and patterns of symmetric cell division in diverse organisms and employed biophysical and mathematical considerations to develop computer simulations that have allowed accurate prediction of cell division patterns. From these studies, a picture emerges that diverse biological systems follow simple universal rules of geometry to select their division planes and that the microtubule cytoskeleton takes a major part in sensing the geometric information and translates this information into a specific division outcome. In plant cells, the division plane is selected before mitosis, and spatial information of the division plane is preserved throughout division by the presence of reference molecules at a distinct region of the plasma membrane, the cortical division zone. The recruitment of these division zone markers occurs multiple times by several mechanisms, suggesting that the cortical division zone is a highly dynamic region. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, the course of the cell cycle depends on correct cytoskeleton arrangement. The cell cycle consists of several phases, and in each of them the cytoskeleton has a unique structure and set of characteristics. The dynamics of the cytoskeleton together with its binding proteins greatly contribute to progression of the cell cycle. Here, we mainly review recent research on the dynamic distribution of the actin cytoskeleton and actin-binding proteins, and the mechanisms by which they affect the progression of the plant cell cycle. [ABSTRACT FROM AUTHOR]

The paper presents previously undescribed abnormalities of phragmoplast formation in pollen mother cells of grass haploids and allohaploids (wide hybrids of the first generation). These abnormal phenotypes reveal and illustrate occasional unknown stages of phragmoplast formation in dividing plant cells and indicate the special regulation of their initiation, as well as confirm data on this process obtained by other approaches. [ABSTRACT FROM AUTHOR]

group of new abnormalities in meiotic division in the mother cells of cereal pollen that affect the processes of the centrifugal movement of a phragmoplast in successive cytokinesis is presented for the first time. These phenotypes represent new information on the formation and operation of motile phragmoplast and confirm our model of centrifugal movement as B-anaphase modification. [ABSTRACT FROM AUTHOR]

The phenomenon of the disorientation of completely formed systemic cytoskeleton structures, i.e., the division spindle and phragmoplast, into the constituent elements and their transformation into a network of disoriented fibers in the course of cell division is described. The phenomenon of the disintegration and dispersion in the cytoplasm of completely formed telophase chromosome groups, which is not associated with the chaotization of the cytoskeleton structures, is also described. These abnormalities are revealed in the meiosis of pollen mother cells of the first generation of wheat-wheatgrass hybrids. The chaotization of cytoskeleton structures is a only normal phenomenon in plant-cell division in late prophase-early prometaphase, whereas, at stages of metaphase and telophase, it can indicate a disturbance in the time regulation of the cytoskeleton cycle in the course of meiotic division. The disintegration of the chromosome telophase groups and their movement backwards to the spindle equator can indicate the untimely involvement of processes of prometaphase, specifically the activation of chromokinesins. The significance of the process of cytoskeleton chaotization in the biology of a plant cell is discussed. [ABSTRACT FROM AUTHOR]

The phragmoplast is a special apparatus that functions in establishing a cell plate in dividing plant cells. It is known that microfilaments (MFs) are involved in constituting phragmoplast structure, but the dynamic distribution and role of phragmoplast MFs are far from being understood. In this study, the precise structure and dynamics of MFs during the initiation and the late lateral expansion of the phragmoplast were observed by using a tobacco BY-2 cell line stably expressing the microfilament reporter construct GFP-fABD2. Three-dimensional imaging showed that the phragmoplast MFs were initiated by two populations of MFs emerging between the reconstituting daughter nuclei at anaphase, which migrated to the mid-zone and gave rise to two layers of microfilament arrays. FM4-64 stained vesicles accumulated and fused with the cell plate between the two populations of MFs. The two layers of microfilament arrays of phragmoplast with ends overlapped always surrounded the centrifugally expanding cell plate. Partial disruption of MFs at metaphase by low concentration of latrunculin B resulted in the inhibition of the cell plate consolidation and the blockage of cell plate lateral expansion, whereas high concentration of latrunculin B restrained the progression of the cell cycle. Treating the cell after the initiation of phragmoplast led to the cease of the expansion of the cell plate. Our observations provide new insights into the precise structure and dynamics of phragmoplast MFs during cytokinesis and suggest that dynamic phragmoplast MFs are important in cell plate formation. [ABSTRACT FROM AUTHOR]

The compensation for phragmoplast dysfunction in the male meiosis of F1 wheat × rye hybrids was described. In pollen mother cells (PMCs), he transition from central spindle fibers (forming a solid bundle) to phragmoplast (hollow cylinder) was blocked. This blockage suppresses the centrifugal movement of the phragmoplast and cell-plate formation. As a result, cells become binucleate. Sometimes, two nuclei fuse and form one restitution nucleus. In PMCs of the wheat × rye F1 hybrid D-144 gp 06 year ( T. aestivum n. 93-60 t 9 × S. cereale n. Saratovskaya 7) with this phenotype, an additional phragmoplast is formed at the late telophase. This occurs by a common mechanism for the development of the immobile phragmoplast in the meiosis in bicotyledons; new phragmoplasts arise as a result of microtubule polymerization starting from the spindle poles. The accessory phragmoplast facilitates a new cell plate assembly and achievement of cytokinesis. [ABSTRACT FROM AUTHOR]

We have studied the response of interphase and mitotic microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) to cold stress (1 h at 0°C) and acclimation to cold (3–48 h at 0°C). We show that, in general, interphase microtubules are more resistant to cold then mitotic arrays in both cultivars. During cold stress, no changes are detected in the microtubule system of interphase cells of spring wheat, whereas the density of endoplasmic microtubules increases in interphase cells of winter wheat. During mitosis, the density of the kinetochore fibers of the spindle decreases in the cells of both cultivars, but it is prevailing in the cells of spring cultivar of wheat. During acclimation to cold, the disorganization of the cortical microtubule bundles and the enhanced growth of the endoplasmic microtubule network, which is comprised of microtubule converging centers, are observed in cells of both cultivars. However, the mitotic microtubule systems of winter and spring cultivars respond differently to cold acclimation. During prophase, a diffuse tubulin “halo,”followed by the assembly of microtubule converging centers, accumulate at the perinuclear area in the cells of winter wheat. In cells of spring cultivar, the prophase spindle is only detected during initial stages of cold acclimation. During metaphase, aberrant mitotic spindles, abnormal metaphase plates, and the excessive appearance of microtubule converging centers are observed in cells of both cultivars. Acclimation induces the disorganization of the phragmoplast and the formation of multiple microtubule converging centers during telophase in the cells of both cultivars. Microtubule converging centers are detected at the perinuclear area of daughter cells in winter wheat and in the cortical cytoplasm in spring wheat. The excessive formation of microtubule converging centers suggests the activation of microtubule assembly during prolonged exposure to low temperature. Our data also demonstrates common pathways of microtubule response to cold treatment (0°C). [ABSTRACT FROM AUTHOR]

During plant cytokinesis, kinesin-related motor proteins are believed to play critical roles in microtubule organization and vesicle transport in the phragmoplast. Previously, we reported that the motor AtPAKRP1 was associated with the plus end of phragmoplast microtubules inArabidopsis thaliana[Lee Y-RJ, Liu B (2000) Curr Biol 10:797-800]. In this paper, we report a full-length cDNA from the same organism, which encodes a polypeptide 74% identical to AtPAKRP1. This AtPAKRP1-like protein-AtPAKRP1L-and AtPAKRP1 share similar domain structures along the polypeptides. Peptide antibodies were raised and purified to distinguish the two polypeptides in vitro and in vivo. When monospecific anti-AtPAKRP1 and anti-AtPAKRP1L antibodies were used in immunofluorescence, they both decorated the plus end of phragmoplast microtubules at all stages of phragmoplast development. Their localization patterns were indistinguishable from each other. By using bacterially expressed fusion proteins of motor-less versions of both polypeptides, it was revealed that AtPAKRP1 and AtPAKRP1L were able to interact with themselves and with each other. Using T-DNA insertional mutants, it was also demonstrated that AtPAKRP1 and AtPAKRP1L were not required for each other’s localization. Our results therefore indicate that AtPAKRP1 and AtPAKRP1L are both expressed in the same cells, and likely have identical functions in the phragmoplast by forming either homodimers or heterodimers. [ABSTRACT FROM AUTHOR]

Organization and rearrangements of microtubular cytoskeleton (MTs) during microsporogenesis are determined in `historical' potato genotypes producing 2 n pollen following different meiotic nuclear restitution mechanisms (`parallel spindles', `fused spindles', `premature cytokinesis'). The scope of the investigation is to assess the abnormalities in MT cytoskeleton and their relationships to 2 n pollen formation. The genotypes of Solanum used for MT immunofluorescence in this study are diploid interspecific hybrids (2n=2 x=24) coming from Wisconsin (USA) and Wageningen (NL) Universities. Based on MT analysis, a reinterpretation of the restitution mechanisms following spindle abnormalities was performed in this study. The fusion of the nuclei at prophase II occurs through the perinuclear MTs in the genotype with `fused spindles'. In genotypes with misoriented spindles, the `non-spindle' cytoskeletal arrays, occurring at telophase II, are clue in determining the meiotic products such as dyads and triads. A particular MT array could be crucially involved in meiotic nuclear restitutions both at telophase I and II: the interzonal MTs. It is discussed which is the relationship between this array and the spindle orientation at meta-anaphase II. It is hypothesized that the fusion of non-sister nuclei at each pole depends on the lack of secondary interzonal MTs and on the short distance between nuclei. In the genotype with premature cytokinesis, effectively, a premature phragmoplast occurs at the end of meiosis I. Furthermore, two genotypes are desynaptic with a high degree of male fertility restored by the nuclear restitutions. However, no clear relationship was found between univalents and restitution mechanisms. [ABSTRACT FROM AUTHOR]

An ethyl methanesulfonate-induced mitotic mutant of durum wheat ( Triticum turgidum L. var. durum; 2 n=4 x=28) was found. We have characterized the mutant to determine the mechanism of abnormal cell division and to test for temperature effects on abnormal cell division. Stained root-tip meristems and pollen mother cells were studied with brightfield, phase contrast, and immunofluorescence microscopy. Abnormal cells included metaphase cells with a multiple of the normal complement (8 x=56, or 16 x=112), multinucleate cells, 4C, 8C, or 16C mononucleate cells, and cells exhibiting incomplete cytokinesis. The mutant had three classes of pollen mother cells: euploid with normal bivalent pairing, multiploid with bivalent pairing, and multiploid with multivalent pairing. Preprophase bands and spindles were normal in mononucleate cells. Some cells had asymmetrical phragmoplasts and phragmoplast dismantling that produced incomplete cytokinesis. Failure of cytokinesis followed by nuclear fusion were the mechanisms of abnormal cell division. To test for temperature sensitivity of the mutant, seedlings were germinated under six different temperature regimes. As germination temperature increased, the frequency of abnormal cells increased. When the mutant was crossed as the female with durum wheat, 3% of hybrids were hexaploid, indicating that functional–unreduced gametes had formed in megaspores. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Microsporocytes of the slipper orchid Cypripedium californicum A. Gray divide simultaneously after second meiosis. The organization and apportionment of the cytoplasm throughout meiosis are functions of nuclear-based radial microtubule systems (RMSs) that define domains of cytoplasm - a single sporocyte domain before meiosis, dyad domains within the undivided cytoplasm after first meiosis, and four spore domains after second meiosis. Organelles migrate to the interface of dyad domains in the undivided cytoplasm after first meiotic division, and second meiotic division takes place simultaneously on both sides of the equatorial organelle band. Microtubules emanating from the telophase II nuclei interact to form columnar arrrays that interconnect all four nuclei, non-sister as well as sister. Cell plates are initiated in these columns of microtubules and expand centrifugally along the interface of opposing RMSs, coalescing in the center of the sporocyte and joining with the original sporocyte wall at the periphery to form the tetrad of microspores. Organelles are distributed into the spore domains in conjunction with RMSs. These data, demonstrating that cytokinesis in microsporogenesis can occur in the absence of both components of the typical cytokinetic apparatus (the preprophase band of microtubules which predicts the division site and the phragmoplast which controls cell-plate deposition), suggest that plant nuclei have an inherent ability to establish a domain of cytoplasm via radial microtubule systems and to regulate wall deposition independently of the more complex cytokinetic apparatus of vegetative cells. [ABSTRACT FROM AUTHOR]

Disagreement has arisen over the presence of actin-containing microfilaments (Mfs) in angiosperm generative cells and sperm (GSP). In order to address this issue, we subjected GSP of Tradescantia virginiana, Nicotiana tabacum and Rhododendron laetum to a series of localizations using different antiactins, rhodamine phalloidin and antimyosin. Coordinate staining with antitubulin and Hoechst 33258 defined the status of the microtubule (Mt) cytoskeleton and stages of generative cell division. Additional experiments utilized cytochalasin D (CD). In no instance could Mfs be detected in GSP of the three species. Instead, Mfs seen at the periphery of GSP appear to be continuous with vegetative Mfs and thus are in the vegetative cytoplasm. Mfs are not seen in the constriction zone of dividing T. virginiana generative cells, nor are they indicated in the phragmoplast of N. tabacum and R. laetum. Myosin localizations reveal punctate staining in the vegetative cytoplasm and a thin line of fluorescence around the the outside of the generative cell. While CD seems to delay generative cell division, cytokinesis still takes place. CD-induced Mf fragments are evident in the vegetative cytoplasm but not in GSP. The weight of evidence therefore indicates that GSP do not contain Mfs. The implications of this conclusion for the behavior of GSP and the mechanism of cytokinesis in dividing generative cells are considerable. [ABSTRACT FROM AUTHOR]

Most bryophytes produce tetrahedral spore tetrads. However, linear spore tetrads have been reported to occur in Conocephalum japonicum (Thunb.) Grolle. In this study, the distribution of microtubules (MTs) during meiosis in C. japonicum was examined to determine the division pattern resulting in a linear tetrad. Spore mother cells in the pre-meiotic stage were cylindrical with randomly distributed cytoplasmic MTs. In the prophase-metaphase transition, spindle MTs replaced cytoplasmic MTs and a barrel-shaped spindle with two flattened poles developed. Cortical MT arrays were not detectable throughout meiosis. Although a phragmoplast appeared between sister nuclei in telophase-I, it disappeared without expanding to the parental cell wall. Metaphase-II spindles oriented parallel to the long axis of the cell and in tandem to each other resulted in a linear arrangement of telophase nuclei. Radial arrays of MTs developed from the nuclear surfaces and three phragmoplasts appeared among the four nuclei to produce four spores. Two phragmoplasts separating the paired sister nuclei appeared prior to the appearance of a phragmoplast between non-sister nuclei. The MT cycle is basically the same as that reported in meiosis of C. conicum, which produces non-linear tetrads. A morphometric study indicated that the difference in the division pattern between C. conicum and C. japonicum is due to a difference in the shape of spore mother cells. The cylindrical shape of sporocytes of C. japonicum restricts the orientation of spindles and phragmoplasts so that the four resultant spores are arranged linearly. [ABSTRACT FROM AUTHOR]

The present work establishes a correlation between cell length and patterns of mitotic microtubular assemblies in Allium cepa L. root meristems. Binucleate cells were formed by a short caffeine treatment which aborted the formation of the phragmoplast during telophase. The largest binucleate cells (about 50 μm in length) behaved as two contiguous mononucleate cells in their next mitosis: they developed two preprophase bands (PPBs), one around each nucleus, where two spindles and two phragmoplasts were subsequently formed. On the other hand, the shortest binucleate cells (about 36 μm in length) formed a single PPB at the site of the aborted phragmoplast and, in the medium-sized cells (about 44 μm) in which the single PPB formed around the nucleus possessing the largest cytoplasmic environment, the two mitotic spindles and the new phragmoplasts moved to, or were assembled in the position of the phragmoplast that had been aborted one cycle earlier. Some rules derive from these observations. First of all, the aborted phragmoplast left a signal for microtubule positioning which was still operative one cycle later, in two-thirds of the bimitoses. Also, that formation of the PPB is dispensable. Moreover, its development does not always predict the future division plane, because of the presence of competing old signals which are stronger than those shed by the PPB in the same mitosis, but which fade away with distance. Finally, the positional signals were reinforced when the ratio of monomeric to fibrillar actin was increased by cytochalasin D during their shedding. When this drug was given simultaneously with caffeine, the frequency of bimitoses which, one cycle later, developed spindles and phragmoplasts in the positions of the old phragmoplast increased. On the other hand, those frequencies dropped in relation to control when the cytochalasin D treatment took place during bimitosis, indicating that at this time the treatment reinforced the signals produced in bimitosis itself. [ABSTRACT FROM AUTHOR]

The organization of the microtubule (Mt) cytoskeleton during mitosis and cytokinesis of the generative cell (GC) in Ornithogalum virens L. (bicellular pollen type, chromosome number, n = 3) from prophase to telophase/sperm formation was investigated by localization of α-tubulin immunofluorescence using a conventional fluorescence microscope and a confocal laser scanning microscope. Chromosomes were visualized with DNA-binding fluorochrome dyes (ethidium bromide and 4′6-diamino-2-phenyl-indole). The GC of O. virens is characterized by G2/M transition within the pollen grain and not in the pollen tube as occurs in the majority of species with bicellular pollen. It was found that prophase in the GC starts before anthesis and prometaphase takes place after 10 min of pollen germination. The prophase Mts are organized into three prominent bundles, located near the generative nucleus. The number of these Mt bundles is the same as the number of GC chromosomes, a relation which has not previously been considered in other species. The most evident feature in the prophase/ prometaphase transition of O. virens GC is a direct rapid rearrangement of Mt bundles into a network which appears to interact with kinetochores and form a typical prometaphase Mt organization. The metaphase chromosomes are arranged into a conventional equatorial plate, and not in tandem as is thought to be characteristic of GC metaphase. The metaphase spindle consists of kinetochore fibres and a few interzonal fibres which form dispersed poles. Anaphase is characterized by a significant elongation of the mitotic spindle concomitant with the extension of the distance between the opposite poles. At anaphase the diffuse poles converge. Cytokinesis is realized by cell plate formation in the equatorial plane of the GC. The phragmoplast Mts between two future sperm nuclei appear after Mts of the mitotic spindle have disappeared. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Caulonema tip cells of Funaria deposit new oblique cross walls of specific morphology and placement by a highly defined reorientation mechanism. In the presence of the purported intracellular Ca antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), these cross walls form in the proper place but exhibit a distorted morphology. Video microscopy indicates that the deformation takes place during the reorientation of the cell plate from a perpendicular to an oblique configuration. Electron micrographs of TMB-8 treated cells indicate a stabilization of phragmoplast microtubules and a greater amount of vesicles and membrane in the developing cell plate. TMB-8 treated cells also show intense chlortetracycline fluorescence from mitochondria, vesicles and endoplasmic reticulum as compared to untreated cells indicating that TMB-8 is blocking release of Ca from intracellular stores. It is concluded that this may cause distortation of cross walls as they form by delaying vesicle fusion, stabilizing microtubules, and increasing the amount of new wall material in the developing cell plate. [ABSTRACT FROM AUTHOR]

The aim of this study was to search for uncharacterized components of the plant cytoskeleton using monoclonal antibodies raised against spermatozoids of the fern Pteridium ( Marc et al. 1988). The cellular distribution of crossreacting immunoreactive material during the division cycle in wheat root tip cells was determined by immunofluorescence microscopy and compared to the fluorescence pattern obtained with antitubulin. Five antibodies are of special interest. Pas1D3 and Pas5F4 detect a diffuse cytoplasmic material, which, during mitosis, follows the distribution of microtubules (MTs) at the nuclear surface and in the preprophase band (PPB), spindle and phragmoplast. The immunoreactive material codistributes specifically with MT arrays of the mitotic apparatus and does not associate with interphase cortical MTs. Pas5D8 is relevant to the PPB and spatial control of cytokinesis. It binds in a thin layer at the cytoplasmic surface throughout the cell cycle, except when its coverage is transiently interrupted by an exclusion zone at the PPB site and later at the same site when the phragmoplast fuses with the parental cell wall. Pas2G6 reacts with a component of basal bodies and the flagellar band in the Pteridium spermatozoid and recognizes irregularly shaped cytoplasmic vesicles in wheat cells. During interphase these particles form a cortical network. Pas6D7 binds to dictyosomes and dictyosome vesicles. At anaphase the vesicles accumulate at the equator and subsequently condense into the cell plate. [ABSTRACT FROM AUTHOR]

The organization of kinetochore fibers was examined in Allium root cells processed for tubulin immunocytochemistry. Metaphase fibers consist of a core or trunk of Mts to which are attached numerous branches, yielding a bottle-brush of fir-tree pattern similar to that reported in Haemanthus endosperm cells. Many of the branches cross the midzone and extend into the opposite half-spindle. In addition, branch Mts associate with more than one kinetochore fiber. During anaphase, branch Mts elongate while the trunks shorten and fuse into polar caps. Our results are discussed in terms of spindle fiber organization and Mt polarity. [ABSTRACT FROM AUTHOR]

Treatment with lysine prior to fixation of tobacco BY-2 cells with formaldehyde improved the preservation of actin filaments in the cells and enabled us to observe both networks of actin filaments and microtubules in the same cells. By using this method, we observed that (1) actin filaments were present in the preprophase band; (2) the actin filaments in the preprophase band and phragmoplast were runnig in the same direction as the microtubules in their respective structures; (3) a cortical network of actin filaments was present throughout all stages of cell cycle. The present method did not preserve the cortical actin filaments in interphase cells. The procedure for staining microtubules destroyed them. [ABSTRACT FROM AUTHOR]

The distribution of F-actin in the phragmoplast/cell plate complex of formaldehyde-fixed Allium root cells was visualized with rhodaminephalloidin (RP). Increased RP fluorescence appears in late anaphase in a broad zone between separating chromosomes. The fluorescence is mostly amorphous in appearance and does not resemble the distinct actin fibers seen in interphase cells. The actin becomes more concentrated near the midplane by telophase and takes the form of a relatively bright layer of fluorescence adjacent to the forming cell plate. This distribution differs markedly from that of phragmoplast microtubules (MTs) which extend back from the plate toward the daughter nuclei. F-actin continues to accumulate in new parts of the expanding phragmoplast, while RP fluorescence gradually decreases near older portions of the plate. It disappears completely near the new wall in most interphase cells. Treatment of root tips with cytochalasin B or D before fixation markedly reduces RP fluorescence, but phragmoplast MTs remain. Colchicine or oryzalin treatment leads to the disappearance of both phragmoplast actin and MTs. The possible function of actin in the phragmoplast/cell plate complex is discussed. [ABSTRACT FROM AUTHOR]

The microtubules (MTs) of developing Sphagnum leaflets rearrange from the interphase array into the preprophase band without obvious participation of definite initiation sites. At late prophase, additional MTs appear along the nuclear envelope, with the same orientation as in the peripherally situated preprophase band. Spindle formation begins along the nuclear envelope; spindle MTs run perpendicular to preprophase band MTs and converge in several focus points with indistinct polar bodies. After cytokinesis, most spindle and phragmoplast MTs disappear. Interphase MTs reappear at first along the central part of the new cell wall, in a region which was occupied before by the initial phragmoplast; their orientation is perpendicular to the phragmoplast MTs. Also here, distinct MT organizing centers could not be observed. Then the MT spread out over the cell periphery. The observations suggest that diffuse MT organizing 'zones' rather than definite MT organizing 'centers' play a role in the rearrangement of the different MT arrays during the cell cycle. [ABSTRACT FROM AUTHOR]

Cytokinesis following asymmetrical pollen mitosis was studied in the slipper orchid Cypripedium fasciculatum using techniques of immunofluorescence, confocal laser scanning, and transmission electron microscopy. Data from stereo reconstructions of double labelled preparations (microtubules/nuclei) show that the contribution of residual spindle fibers to development of the interzonal array is minor; rather, new populations of microtubules are nucleated in association with the two groups of anaphase chromosomes. As kinetochores reach the poles, trailing arms of the chromosomes and nonkinetochore microtubules are displaced outward in the equatorial zone and by early telophase the interzone is left virtually free of microtubules. The interzonal apparatus has its origin in a massive proliferation of microtubules from the polar regions and surfaces of contracting chromosomes. Each polar region appears as a hub from which microtubules radiate in a spoke-like configuration and numerous tufts of microtubules appear to emanate from margins of the chromosomes themselves. These newly organized arrays of microtubules extend to the equatorial region where they interact to form the interzonal apparatus. Increasing organization of microtubules in the interzone results in development of a typical phragmoplast configuration consisting of opposing cone-like bundles of microtubules bisected by an unstained equatorial line. [ABSTRACT FROM AUTHOR]

The distribution of microtubules and actin microfilaments during caffeine-induced inhibition of cell plate formation has been studied in living Tradescantia 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. [ABSTRACT FROM AUTHOR]

We have obtained immunofluorescence and immunoblot evidence for the presence of kinesin-like protein (KLP) in pollen tubes of tobacco using an antibody generated against peptides encoded by the KATA gene of Arabidopsis. This antibody recognizes an M 140,000 polypeptide in Arabidopsis seedlings, and stains the mitotic apparatus in this species as well as in tobacco suspension cells. In tobacco pollen tubes prepared for dual immunofluorescence localizations of KLP and β-tubulin, the antibody binds transiently to microtubule (Mt) bundles and the nucleus in premitotic generative cells; it then stains the developing mitotic apparatus as the nuclear envelope breaks down. By metaphase, fluorescence is located over kinetochore fibers and associated Mts. Localization of KLP is concentrated in the midzone during anaphase, and by early cytokinesis, it closely brackets the cell plate. Phragmoplast fluorescence then spreads along the phragmoplast distal to the cell plate. Punctate staining is also detected along vegetative Mts. No KLP localization is seen in pollen tubes treated with antibody after it had been preadsorbed to the antigenic peptides. The antibody recognizes an M 110,000 polypeptide in extracts of tobacco pollen tubes, and a polypeptide of somewhat lower M in Tradescantia pollen tubes. Our results show that KLP(s) related to KatAp are present in tobacco generative cells and may play roles in the organization and/or operation of the mitotic apparatus and phragmoplast. [ABSTRACT FROM AUTHOR]

The structure of the mitotic apparatus during the microspore division of Tradescantia paludosa, which has a distinctively unequal division of large vegetative and small generative cells, was studied using α-tubulin immunofluorescence methods and confocal laser scanning microscopy. Mitotic apparatuses began to develop asynchronously during early prophase at the vegetative pole (VP) and during prometaphase at the generative pole (GP). Both, however, reached completion together at the same time during metaphase. At the VP from prophase to prometaphase, microtubules (MTs) did not converge on the pole, and there was a circular area containing only a few MTs. The prophase spindles on the VP side were in the form of domes or cones that lacked the top. In the metaphase, however, the MTs concentrated at the pole to form a representative cone-shaped half-spindle. At the GP from prometaphase to metaphase, the MTs did not concentrate, and a circular area existed that lacked MTs. The half-spindles formed truncated cones. When the phragmoplast developed and curved around the generative nucleus during the telophase. it first grew toward the long axis of the ellipsoidal-shaped microspore; and after it arrived at the inner membrane of the microspore, it again curved past the generative nucleus toward the short axis. In conclusion, it was found that the mitotic apparatus of T. paludosa microspores with its asynchronous growth and asymmetrical spindle structure and with its three dimensional growth of phragmoplasts had a peculiar developmental manner related to unequal division. [ABSTRACT FROM AUTHOR]

Although there are numerous herbicides that disrupt mitosis as a mechanism of action, to date not one has compared the effects of these disrupters on a single species and over a range of concentrations. Oat seedlings, treated with a range of concentrations of nine different 'mitotic disrupter herbicides', were examined by immunofluorescence microscopy of tubulin in methacrylate sections. All herbicides caused the same kinds of microtubule disruption, although the concentrations required to cause the effects differed markedly between the herbicides. Effects on spindle and phragmoplast mitotic microtubule arrays were seen at the lowest concentrations and manifested as multipolar spindles and bifurcated phragmoplasts (which subsequently resulted in abnormal cell plate formation). At increasing concentrations, effects on mitotic microtubule arrays manifested as microtubule tufts at kinetochores and reduction of cortical microtubules resulting in arrested prometaphase figures and isodiametric cells. These data indicate that all mitotic disrupter herbicides have a common primary mechanism of action, inhibition of microtubule polymerization, and that marginal effects observed in the past were the result of incomplete inhibition and/or differential sensitivity of the microtubule arrays. [ABSTRACT FROM AUTHOR]

In order to gain a more complete understanding of the organization of the mitotic apparatus (MA) in the generative cells (GCs) of flowering plants, pollen tubes of Nicotiana tabacum were examined using tubulin immunocytochemistry and Hoechst fluorescence. The observations were then compared with previously published information on Tradescantia GCs and the MA of somatic cells. At the onset of division, the prominent microtubule (Mt) bundles characteristic of GCs are reorganized into a more random Mt network. At late prophase/prometaphase, kinetochores appear to interact with this network, resulting in the formation of K-fibers that frequently link in tree-like aggregates. The GC MA takes the form of a distinct spindle and often has pointed, focused poles; the metaphase plate is usually oblique. Karyokinesis involves both anaphase A and B; lengthening of interzonal Mts is accompanied by elongation of the spindle. In late anaphase/early telophase, phragmoplast Mts are formed in association with the proximal face of the sperm nuclei. The phragmoplast remains prominent for some time, so that its Mts as well as another population generated from the distal face of the sperm nuclei constitute the initial sperm cytoskeleton. Comparisons indicate that the spindle in tobacco GCs falls on a continuum of organization between that of somatic cells and the MA of Tradescantia GCs. [ABSTRACT FROM AUTHOR]

Cytokinesis in microsporocytes of moth orchids is unusual in that it occurs simultaneously after meiosis, the cytoplasm does not infurrow in the division planes, and cell plates are deposited in association with centrifugal expansion of phragmoplasts. Microtubules radiating from the nuclear envelopes appear to be of fundamental importance in establishment of division planes. Primary interzonal spindles develop between sister nuclei and interaction of radial microtubules triggers development of secondary interzonal spindles between non-sister nuclei. From three to six or more phragmoplasts, depending upon the arrangement of nuclei in the coenocyte, develop from these postmeiotic arrays. The phragmoplasts consist of co-aligned microtubules and F-actin organized into bundles that are broad proximal to the mid-plane and taper distally. Ultrastructure of the phragmoplast/cell plate reveals that abundant ER is associated with vesicle aggregation and coalescence. Cell plates are deposited in association with phragmoplasts as they expand centrifugally to join the parental wall and/or fuse with one another in the interior of the cell. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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