Accumulation of F-actin during cytokinesis inAllium. Correlation with microtubule distribution and the effects of drugs

Summary The distribution of F-actin in the phragmoplast/cell plate complex of formaldehyde-fixedAllium 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.Abbreviations CB cytochalasin B - CD cytochalasin D - CIPC isopropyl N-(3-chlorophenyl-)carbamate - DIC differential interference contrast - MT microtubule - PBS phosphate buffered saline - PM plasmalemma - RP rhodamine-phalloidin     

Dynamics of microtubular cytoskeleton in higher plant meiosis. VII. Mechanisms of the simultaneous cytokinesis

Rearrangements of microtubular cytoskeleton during telophase in pollen mother cells of some dicotyledon plants with the simultaneous cytokinesis during normal and abnormal meiosis were studied. At telophase I, a potentially functional phragmoplast forms between daughter nuclei, but no cell plate is present. During interkinesis, the phragmoplast plays the role of an interphase cytoskeleton array. Dynamics of microtubule reorganization in polar regions of the telophase spindle is discussed in addition to the role played by microtubule convergence centers in cytoskeleton rearrangements during meiosis.     

CELL DIVISION IN SPIROGYRA. II. CYTOKINESIS

Cytokinesis in cells of Spirogyra sp. was studied with both light and electron microscopes. Early formation of the cross wall was achieved by annular ingrowth of a septum; the cross wall was completed by a phragmoplast containing Golgi vesicles, longitudinally aligned microtubules, and associated electron-dense material. Spirogyra may represent an intermediate stage in the evolution of the phragmoplast seen in higher plants.     

Inhibition of cell-plate formation by brefeldin A inhibited the depolymerization of microtubules in the central region of the phragmoplast

Treatment of tobacco BY-2 cells with 20 microM brefeldin A (BFA), which causes disassembly of the Golgi apparatus (Yasuhara et al. 1995), completely inhibited the formation of the cell plate when the treatment was started before the chromosomes had begun to to condense. In cells in which cell-plate formation was inhibited by BFA, the centrifugal development of the phragmoplast was also inhibited. In such cells, the depolymerization of microtubules in the central region of the phragmoplast did not occur at least for 1 h after the formation of the phragmoplast, while the centrifugal development of the phragmoplast and cell-plate formation were completed in almost all cells not treated with BFA. The inhibition of cell-plate formation seems to inhibit the centrifugal development of the phragmoplast by inhibiting the depolymerization of microtubules in the central region of the phragmoplast, which is required for the supply of free tubulin necessary for the polymerization of microtubules at the outer margins of the phragmoplast.     

Effects of Taxol on the Development of the Cell Plate and of the Phragmoplast in Tobacco BY-2 Cells

To ascertain whether accumulation of vesicles at the site ofcell-plate formation in the phragmoplast is caused by the translocationof vesicles along phragmoplast microtubules or by the translocationof vesicles that is mediated by depolymerization of phragmoplastmicrotubules at the equatorial plane, we examined the effectsof taxol, an inhibitor of the depolymerization of microtubules,on the accumulation of vesicles at the equatorial region ofthe phragmoplast in tobacco BY-2 cells. Taxol caused an increase in the accumulation of vesicles atthe equatorial plane of the phragmoplast while simultaneouslyinhibiting the centrifugal growth of the phragmoplast. The depolymerization of microtubules does not seem to be involvedin the accumulation of vesicles at the site of cell-plate formation,but this process appears to be required for the centrifugalgrowth of the phragmoplast. Our results suggest that the translocationof vesicles is mediated by some kind of translocator, whichmoves along phragmoplast microtubules, and that the polymerizationof microtubules at the growing edges of the phragmoplast requiresa supply of free tubulin from preexisting microtubules. (Received May 14, 1992; Accepted October 12, 1992)     

Dynamics of microtubular cytoskeleton in higher plant meiosis. IX. The cycle accomplishment. Transition from phragmoplast to radial microtubule system

In this study we analysed the terminal step of cytoskeleton cycle in higher plant meiosis: transition from phragmoplast to radial interphase configuration. Wild type meiosis in a range of mono- and dicotyledonous species was studied. A number of cytoskeleton abnormalities on this stage was described in meiotic mutants, haploids and wide hybrids of various species. We described processes of cytoskeleton rearrangements on this stage: disjunction of phragmoplast MTs, their shortening and the role of daughter cell membranes. The independence of the interphase radial MT system formation from the previous steps of cytoskeleton cycle and from nuclear envelope cycle is proposed.     

Altering of phragmoplast orientation as a result of excessive centrifugal movement in the absence of cell plate

The inability of phragmoplast to stop its centrifugal movement after reaching the mother cell membrane is described in abnormal meiosis with the arrest of cell plate formation. The excess of phragmoplast expansion leads to rotation of the whole telophase figure (phragmoplast with daughter nuclei) within the cell through 90 degrees. It has been suggested that this phenomenon may occur because of a the lack of signal stopping cytokinesis. Such a signal arises due to formation of daughter cell membranes.     

A MAP kinase is activated late in plant mitosis and becomes localized to the plane of cell division.

In eukaryotes, mitogen-activated protein kinases (MAPKs) are part of signaling modules that transmit diverse stimuli, such as mitogens, developmental cues, or various stresses. Here, we report a novel alfalfa MAPK, Medicago MAP kinase 3 (MMK3). Using an MMK3-specific antibody, we detected the MMK3 protein and its associated activity only in dividing cells. The MMK3 protein could be found during all stages of the cell cycle, but its protein kinase activity was transient in mitosis and correlated with the timing of phragmoplast formation. Depolymerization of microtubules by short treatments with the drug amiprophosmethyl during anaphase and telophase abolished MMK3 activity, indicating that intact microtubules are required for MMK3 activation. During anaphase, MMK3 was found to be concentrated in between the segregating chromosomes; later, it localized at the midplane of cell division in the phragmoplast. As the phragmoplast microtubules were redistributed from the center to the periphery during telophase, MMK3 still localized to the whole plane of division; thus, phragmoplast microtubules are not required to keep MMK3 at this location. Together, these data strongly support a role for MMK3 in the regulation of plant cytokinesis.     

Dynamics of spindle fibers and microtubules during anaphase and phragmoplast formation

Detailed correlation of in vitro observations with the arrangement of microtubules (MTs) during anaphase-telophase were made on endosperm of Haemanthus katherinae. It is stressed that the general course of events leading to the formation of the phragmoplast is the same in all cells, but considerable variation of details may be found in different objects and even in various cells of the same tissue. The changes of MT arrangement in the interzonal region responsible for formation of the phragmoplast already occur in anaphase. During this stage continuous fibers (composed of numerous MTs) lengthen, become thinner (the number of MTs on a cross-section decreases), and often seem to break. After mid-anaphase, thin fibers begin to oscillate transversely to the axis of the phragmoplast and often are considerably laterally displaced (lateral movements). The longest MTs in the phragmoplast are present during oscillations and lateral movements. The new MTs arise in the phragmoplast regions depleted of MTs as a result of lateral movements (usually geometric central region of the phragmoplast). Clusters of vesicles, which accumulate in relation to MTs which move, fuse and form the cell plate. After the fusion, the number and the length of MTs decrease. Several processes are superimposed and occur simultaneously. Also the cell plate is, as a rule, in different stages of development in various regions of the phragmoplast. The movements of MTs and fusion of the vesicles is complex and the details of these processes are not entirely clear. The data supplied here modify some generally accepted concepts of phragmoplast formation and development. This concerns the center of origin of new MTs, the moment when they arise, and the way they subsequently behave.     

Electron tomographic analysis of somatic cell plate formation in meristematic cells of Arabidopsis preserved by high-pressure freezing

We have investigated the process of somatic-type cytokinesis in Arabidopsis (Arabidopsis thaliana) meristem cells with a three-dimensional resolution of approximately 7 nm by electron tomography of high-pressure frozen/freeze-substituted samples. Our data demonstrate that this process can be divided into four phases: phragmoplast initials, solid phragmoplast, transitional phragmoplast, and ring-shaped phragmoplast. Phragmoplast initials arise from clusters of polar microtubules (MTs) during late anaphase. At their equatorial planes, cell plate assembly sites are formed, consisting of a filamentous ribosome-excluding cell plate assembly matrix (CPAM) and Golgi-derived vesicles. The CPAM, which is found only around growing cell plate regions, is suggested to be responsible for regulating cell plate growth. Virtually all phragmoplast MTs terminate inside the CPAM. This association directs vesicles to the CPAM and thereby to the growing cell plate. Cell plate formation within the CPAM appears to be initiated by the tethering of vesicles by exocyst-like complexes. After vesicle fusion, hourglass-shaped vesicle intermediates are stretched to dumbbells by a mechanism that appears to involve the expansion of dynamin-like springs. This stretching process reduces vesicle volume by approximately 50%. At the same time, the lateral expansion of the phragmoplast initials and their CPAMs gives rise to the solid phragmoplast. Later arriving vesicles begin to fuse to the bulbous ends of the dumbbells, giving rise to the tubulo-vesicular membrane network (TVN). During the transitional phragmoplast stage, the CPAM and MTs disassemble and then reform in a peripheral ring phragmoplast configuration. This creates the centrifugally expanding peripheral cell plate growth zone, which leads to cell plate fusion with the cell wall. Simultaneously, the central TVN begins to mature into a tubular network, and ultimately into a planar fenestrated sheet (PFS), through the removal of membrane via clathrin-coated vesicles and by callose synthesis. Small secondary CPAMs with attached MTs arise de novo over remaining large fenestrae to focus local growth to these regions. When all of the fenestrae are closed, the new cell wall is complete. Few endoplasmic reticulum (ER) membranes are seen associated with the phragmoplast initials and with the TVN cell plate that is formed within the solid phragmoplast. ER progressively accumulates thereafter, reaching a maximum during the late PFS stage, when most cell plate growth is completed.     

Caffeine inhibits callose deposition in the cell plate and the depolymerization of microtubules in the central region of the phragmoplast

Treatment of tobacco BY-2 cells with 10 mM caffeine that was started after the cells had entered the mitotic phase did not completely inhibit the deposition of callose in the cell plate and allowed the centrifugal redistribution of phragmoplast microtubules. On the other hand, when treatment with caffeine was started before the cells entered the mitotic phase, the deposition of callose was completely inhibited and the redistribution of phragmoplast microtubules was also inhibited. As the inhibition of redistribution of phragmoplast microtubules seems to be caused by the inhibition of depolymerization of microtubules at the central region of the phragmoplast, these results strongly suggest that the deposition of callose in the cell plate is tightly linked with the depolymerization of phragmoplast microtubules. Callose deposition was observed in phragmoplasts isolated from caffeine-treated cells as well as in those isolated from non-caffeine-treated cells, and caffeine did not inhibit callose synthesis in isolated phragmoplast, indicating that caffeine neither inhibits the accumulation of callose synthase at the equatorial regions of the phragmoplast nor arrests callose synthase itself.     

AtMAC stabilizes the phragmoplast by crosslinking microtubules and actin filaments during cytokinesis

The phragmoplast, a structure crucial for the completion of cytokinesis in plant cells, is composed of antiparallel microtubules (MTs) and actin filaments (AFs). However, how the parallel structure of phragmoplast MTs and AFs is maintained, especially during centrifugal phragmoplast expansion, remains elusive. Here, we analyzed a new Arabidopsis thaliana MT and AF crosslinking protein (AtMAC). When AtMAC was deleted, the phragmoplast showed disintegrity during centrifugal expansion, and the resulting phragmoplast fragmentation led to incomplete cell plates. Overexpression of AtMAC increased the resistance of phragmoplasts to depolymerization and caused the formation of additional phragmoplasts during cytokinesis. Biochemical experiments showed that AtMAC crosslinked MTs and AFs in vitro, and the truncated AtMAC protein, N-CC1, was the key domain controlling the ability of AtMAC. Further analysis showed that N-CC1(51–154) is the key domain for binding MTs, and N-CC1(51–125) for binding AFs. In conclusion, AtMAC is the novel MT and AF crosslinking protein found to be involved in regulation of phragmoplast organization during centrifugal phragmoplast expansion, which is required for complete cytokinesis.     

Development and disintegration of phragmoplasts in living cultured cells of a GFP::TUA6 transgenic Arabidopsis thaliana plant

Summary.  Cultured suspension cells of Arabidopsis thaliana that stably express a green-fluorescent protein–α-tubulin 6 fusion protein were used to follow the development and disintegration of phragmoplasts. The development and disintegration of phragmoplasts in the living cultured cells could be successively observed by detecting the green-fluorescent protein fluorescence of the microtubules. In the early telophase spindle, where two kinetochore groups and two daughter chromosome groups had completely separated from one another, fluorescence appeared in the interzone between the two chromosome groups. The fluorescent region was gradually condensed at the previous equator and increased in fluorescence intensity, and finally it formed the initial phragmoplast. The initial phragmoplast moved from the cell center towards the cell periphery, and it lost fluorescence at its center and became double rings in shape. The expansion orientation of the phragmoplast was not always the same as that of the future new cell wall before it came in contact with the cell wall. The phragmoplast did not usually come in contact with the cell wall simultaneously with its entire length. A portion of the phragmoplast which was earlier in contact with the cell wall disappeared earlier than other portions of the phragmoplast. The duration of contact between any portions of the phragmoplast and the plasma membrane of the cell wall was 15–30 min. The fluorescence intensity of the cytoplasm did not seem to be elevated by the disintegration of the strongly fluorescent phragmoplast. Received August 8, 2002; accepted September 25, 2002; published online March 11, 2003     

Accessory phragmoplast corrects abnormal cytokinesis in wheat x rye hybrids

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.     

Interaction of antiparallel microtubules in the phragmoplast is mediated by the microtubule-associated protein MAP65-3 in Arabidopsis

In plant cells, microtubules (MTs) in the cytokinetic apparatus phragmoplast exhibit an antiparallel array and transport Golgi-derived vesicles toward MT plus ends located at or near the division site. By transmission electron microscopy, we observed that certain antiparallel phragmoplast MTs overlapped and were bridged by electron-dense materials in Arabidopsis thaliana. Robust MT polymerization, reported by fluorescently tagged End Binding1c (EB1c), took place in the phragmoplast midline. The engagement of antiparallel MTs in the central spindle and phragmoplast was largely abolished in mutant cells lacking the MT-associated protein, MAP65-3. We found that endogenous MAP65-3 was selectively detected on the middle segments of the central spindle MTs at late anaphase. When MTs exhibited a bipolar appearance with their plus ends placed in the middle, MAP65-3 exclusively decorated the phragmoplast midline. A bacterially expressed MAP65-3 protein was able to establish the interdigitation of MTs in vitro. MAP65-3 interacted with antiparallel microtubules before motor Kinesin-12 did during the establishment of the phragmoplast MT array. Thus, MAP65-3 selectively cross-linked interdigitating MTs (IMTs) to allow antiparallel MTs to be closely engaged in the phragmoplast. Although the presence of IMTs was not essential for vesicle trafficking, they were required for the phragmoplast-specific motors Kinesin-12 and Phragmoplast-Associated Kinesin-Related Protein2 to interact with MT plus ends. In conclusion, we suggest that the phragmoplast contains IMTs and highly dynamic noninterdigitating MTs, which work in concert to bring about cytokinesis in plant cells.     

洋葱小孢子母细胞减数分裂过程中微管分布变化

应用间接免疫荧光标记技术和激光共聚焦扫描显微镜成像技术观察洋葱小孢子母细胞减数分裂过程中微管分布变化。减数分裂之前,小孢子母细胞中的微管较短,呈辐射状,由细胞核表面向四周扩散。减数分裂开始后,细胞质中的一部分微管蛋白聚集成纺锤体微管,控制染色体的分布。进入减数分裂I后期,纺锤体微管变为牵引染色体移向两极的着丝粒微管和连接纺锤体两极的极丝微管。之后,所有微管集中在两个核之间,构成成膜体。然后,微管解聚成微管蛋白弥散在细胞质中。减数分裂I完成后,二分体2个子细胞中的微管蛋白又聚集成2个纺锤体微管,开始减数分裂II过程。经过减数分裂II中期,2个二分体细胞中的微管再次集中在2个细胞核之间形成成膜体,隔离2个细胞核。此后,微管蛋白解聚,弥散分布在小孢子细胞质中。     

Phragmoplastin, a dynamin-like protein associated with cell plate formation in plants.

Cytokinesis in a plant cell is accomplished by the formation of a cell plate in the center of the phragmoplast. Little is known of the molecular events associated with this process. In this study, we report the identification of a dynamin-like protein from soybean and demonstrate that this protein is associated with the formation of the cell plate. Plant dynamin-like (PDL) protein contains 610 amino acids showing high homology with other members of the dynamin protein family. Western blot experiments demonstrated that it is associated with the non-ionic detergent-resistant fraction of membranes. Indirect immunofluorescence microscopy localized PDL to the cell plate in dividing soybean root tip cells. Double labeling experiments demonstrated that, unlike phragmoplast microtubules which are concentrated on the periphery of the forming plate, PDL is located across the whole width of the newly formed cell plate. Based on the temporal and spatial organization of PDL in the phragmoplast, we termed this protein 'phragmoplastin'. The data suggest that phragmoplastin may be associated with exocytic vesicles that are depositing cell plate material during cytokinesis in the plant cell.     

A Novel Plant Kinesin-Related Protein Specifically Associates with the Phragmoplast Organelles

In higher plants, the formation of the cell plate during cytokinesis requires coordinated microtubule (MT) reorganization and vesicle transport in the phragmoplast. MT-based kinesin motors are important players in both processes. To understand the mechanisms underlying plant cytokinesis, we have identified AtPAKRP2 (for Arabidopsis thaliana phragmoplast-associated kinesin-related protein 2). AtPAKRP2 is an ungrouped N-terminal motor kinesin. It first appeared in a punctate pattern among interzonal MTs during late anaphase. When the phragmoplast MT array appeared in a mirror pair, AtPAKRP2 became more concentrated near the division site, and additional signal could be detected elsewhere in the phragmoplast. In contrast, the previously identified AtPAKRP1 protein is associated specifically with bundles of MTs in the phragmoplast at or near their plus ends. Localization of the tobacco homolog(s) of AtPAKRP2 was altered by treatment of brefeldin A in BY-2 cells. We discuss the possibility that AtPAKRP1 plays a role in establishing and/or maintaining the phragmoplast MT array, and AtPAKRP2 may contribute to the transport of Golgi-derived vesicles in the phragmoplast.     

Expansion of the phragmoplast during plant cytokinesis: a MAPK pathway may MAP it out

Plant cytokinesis involves the formation of a cell plate. This is accomplished with the help of the phragmoplast, a plant-specific cytokinetic apparatus that consists of microtubules and microfilaments. During centrifugal growth of the cell plate, the phragmoplast expands to keep its microtubules at the leading edge of the cell plate. Recent studies have revealed potential regulators of phragmoplast microtubule dynamics and the involvement of a mitogen-activated protein kinase cascade in the control of phragmoplast expansion. These studies provide new insights into the molecular mechanisms of plant cytokinesis.     

The origin of phragmoplast asymmetry

The phragmoplast coordinates cytokinesis in plants [1]. It directs vesicles to the midzone, the site where they coalesce to form the new cell plate. Failure in phragmoplast function results in aborted or incomplete cytokinesis leading to embryo lethality, morphological defects, or multinucleate cells [2, 3]. The asymmetry of vesicular traffic is regulated by microtubules [1, 4, 5, 6], and the current model suggests that this asymmetry is established and maintained through treadmilling of parallel microtubules. However, we have analyzed the behavior of microtubules in the phragmoplast using live-cell imaging coupled with mathematical modeling and dynamic simulations and report that microtubules initiate randomly in the phragmoplast and that the majority exhibit dynamic instability with higher turnover rates nearer to the midzone. The directional transport of vesicles is possible because the majority of the microtubules polymerize toward the midzone. Here, we propose the first inclusive model where microtubule dynamics and phragmoplast asymmetry are consistent with the localization and activity of proteins known to regulate microtubule assembly and disassembly.