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)     

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.     

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.     

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.     

Two Arabidopsis phragmoplast-associated kinesins play a critical role in cytokinesis during male gametogenesis

In plant cells, cytokinesis is brought about by the phragmoplast. The phragmoplast has a dynamic microtubule array of two mirrored sets of microtubules, which are aligned perpendicularly to the division plane with their plus ends located at the division site. It is not well understood how the phragmoplast microtubule array is organized. In Arabidopsis thaliana, two homologous microtubule motor kinesins, PAKRP1/Kinesin-12A and PAKRP1L/Kinesin-12B, localize exclusively at the juxtaposing plus ends of the antiparallel microtubules in the middle region of the phragmoplast. When either kinesin was knocked out by T-DNA insertions, mutant plants did not show a noticeable defect. However, in the absence of both kinesins, postmeiotic development of the male gametophyte was severely inhibited. In dividing microspores of the double mutant, microtubules often became disorganized following chromatid segregation and failed to form an antiparallel microtubule array between reforming nuclei. Consequently, the first postmeiotic cytokinesis was abolished without the formation of a cell plate, which led to failures in the birth of the generative cell and, subsequently, the sperm. Thus, our results indicate that Kinesin-12A and Kinesin-12B jointly play a critical role in the organization of phragmoplast microtubules during cytokinesis in the microspore that is essential for cell plate formation. Furthermore, we conclude that Kinesin-12 members serve as dynamic linkers of the plus ends of antiparallel microtubules in the phragmoplast.     

Cytoskeletal dynamics in living plant cells

Microtubules and microfilaments have been imaged in living plant cells and their dynamic changes recorded during division, growth and development. Carboxyfluorescein labeled brain tubulin has been injected into cells that are maintained in an active state in a culture chamber on the microscope stage. Subsequent imaging with the confocal microscope reveals microtubules in the preprophase band, the mitotic apparatus, the phragmoplast, and the cortical array. The structural changes of these microtubules have been observed during transitional stages. In addition, their dynamic features are demonstrated by depolymerization in elevated calcium, low temperature, and in the drug oryzalin, and by repolymerization when returned to normal conditions. Examination of living Tradescantia stamen hair cells, which have been injected with fluorescent phalloidin to label the actin microfilaments, reveals hitherto undisclosed aspects of the preparation of the division site and dynamics of the phragmoplast cytoskeleton. During prophase microfilaments occur throughout the cell cortex, with those in the region of the preprophase band becoming transversely aligned. At nuclear envelope breakdown, these specifically disassemble, leaving a circumferential zone from which microfilaments remain absent throughout division. During cytokinesis microfilaments arise within the phragmoplast, oriented parallel to the microtubules, but excluded from the zone where the MTs overlap and where cell plate vesicles aggregate. The phragmoplast microfilaments, in a manner similar to microtubules, shorten in length, expand in girth, and eventually disassemble when the cell plate is complete.     

Completion of cytokinesis in C. elegans requires a brefeldin A-sensitive membrane accumulation at the cleavage furrow apex

BACKGROUND: The terminal phase of cytokinesis in eukaryotic cells involves breakage of the intercellular canal containing the spindle midzone and resealing of the daughter cells. Recent observations suggest that the spindle midzone is required for this process. In this study, we investigated the possibility that targeted secretion in the vicinity of the spindle midzone is required for the execution of the terminal phase of cytokinesis. RESULTS: We inhibited secretion in early C. elegans embryos by treatment with brefeldin A (BFA). Using 4D recordings of dividing cells, we showed that BFA induced stereotyped failures in the terminal phase of cytokinesis; although the furrow ingressed normally, after a few minutes the furrow completely regressed, even though spindle midzone and midbody microtubules appeared normal. In addition, using an FM1-43 membrane probe, we found that membrane accumulated locally at the apices of the late cleavage furrows that form the persisting intercellular canals between daughter cells. However, in BFA-treated embryos this membrane accumulation did not occur, which possibly accounts for the observed cleavage failures. CONCLUSIONS: We have shown that BFA disrupts the terminal phase of cytokinesis in the embryonic blastomeres of C. elegans. We observed that membrane accumulates at the apices of the late cleavage furrow by means of a BFA-sensitive mechanism. We suggest that this local membrane accumulation is necessary for the completion of cytokinesis and speculate that the spindle midzone region of animal cells is functionally equivalent to the phragmoplast of plants and acts to target secretion to the equatorial plane of a cleaving cell.     

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.     

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.     

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.     

Effect of brefeldin A and the dynamics of the actin plate on cytokinesis of zygotes in the brown alga,Silvetia babingtonii (Fucales,Phaeophyceae)

In the cytokinesis of brown algae, actin filaments appear like a plate at the intersecting region of microtubules (MTs) that emerge from the centrosomes after mitosis. The function of the actin plate itself is still unknown. To elucidate the relationship between the actin plate, MTs and membrane fusion, without inducing cytoskeleton depolymerization, the effect of brefeldin A (BFA), which prevents the production of vesicles from Golgi bodies, was examined in zygotes of Silvetia babingtonii. The beginning of mitosis was slightly delayed in zygotes under BFA compared with the controls. Almost all zygotes were inhibited for the progression of cytokinesis by BFA treatment. Ultrastructural observations showed that Golgi cisternae became fragmented or curled following continuous treatment with BFA, and the inhibitory status of cytokinesis between zygotes. The next cell cycle started before cytokinesis was completed. Although the appearance of the actin plate was not disturbed by BFA treatment, the behaviour of the actin plate during the transition between the first and second cell cycles could be classified into two patterns: it was either invisible upon the initiation of the next cell cycle, or a portion of it remained even though the next cell cycle had begun. In the latter case, a part of the actin plate seemed to associate with the new partially formed cell partition membrane, and MTs from the centrosomes were bound to it. The actin plate completely disappeared in the next mitosis, then re-emerged in the middle area of the four daughter nuclei. The results of the present study indicated that, under BFA treatment, the actin plate persisted until just before the beginning of the next mitotic phase, when the new, incomplete cell partition membrane was present, and MTs sustained the actin plate until next mitosis.     

The Arabidopsis HINKEL gene encodes a kinesin-related protein involved in cytokinesis and is expressed in a cell cycle-dependent manner

Plant cytokinesis starts in the center of the division plane, with vesicle fusion generating a new membrane compartment, the cell plate, that subsequently expands laterally by continuous fusion of newly arriving vesicles to its margin. Targeted delivery of vesicles is assisted by the dynamic reorganization of a plant-specific cytoskeletal array, the phragmoplast, from a solid cylinder into an expanding ring-shaped structure. This lateral translocation is brought about by depolymerization of microtubules in the center, giving way to the expanding cell plate, and polymerization of microtubules along the edge. Whereas several components are known to mediate cytokinetic vesicle fusion [8-10], no gene function involved in phragmoplast dynamics has been identified by mutation. Mutations in the Arabidopsis HINKEL gene cause cytokinesis defects, such as enlarged cells with incomplete cell walls and multiple nuclei. Proper targeting of the cytokinesis-specific syntaxin KNOLLE [8] and lateral expansion of the phragmoplast are not affected. However, the phragmoplast microtubules appear to persist in the center, where vesicle fusion should result in cell plate formation. Molecular analysis reveals that the HINKEL gene encodes a plant-specific kinesin-related protein with a putative N-terminal motor domain and is expressed in a cell cycle-dependent manner similar to the KNOLLE gene. Our results suggest that HINKEL plays a role in the reorganization of phragmoplast microtubules during cell plate formation.     

Ultrastructure of cell division in the fusiform cells of the vascular cambium of Robinia pseudoacacia

 The ultrastructure of periclinally dividing fusiform cells was studied in the vascular cambium of Robinia pseudoacacia. Fusiform cell division begins in April at Madison, Wisconsin, when the cambial cells still have many characteristics of a dormant cambium. Soon afterward, the cambial cells acquire the appearance typical of an active cambium. Sequential phases of the microtubule cycle were documented: cortical microtubules bordering the cell wall during interphase, perinuclear microtubules preceding formation of the mitotic spindle, spindle microtubules, and phragmoplast microtubules. A preprophase band of microtubules was not encountered. An extended phragmosome was not encountered in periclinally dividing fusiform cells. During cytokinesis, the phragmosome is represented by a broad cytoplasmic plate which precedes the developing phragmoplast and cell plate as they migrate toward the ends of the cell.     

The cytoplast concept in dividing plant cells: cytoplasmic domains and the evolution of spatially organized cell

The unique cytokinetic apparatus of higher plant cells comprises two cytoskeletal systems: a predictive preprophase band of microtubules (MTs), which defines the future division site, and the phragmoplast, which mediates crosswall formation after mitosis. We review features of plant cell division in an evolutionary context and from the viewpoint that the cell is a domain of cytoplasm (cytoplast) organized around the nucleus by a cytoskeleton consisting of a single "tensegral" unit. The term "tensegrity" is a contraction of "tensional integrity" and the concept proposes that the whole cell is organized by an integrated cytoskeleton of tension elements (e.g., actin fibers) extended over compression-resistant elements (e.g., MTs).During cell division, a primary role of the spindle is seen as generating two cytoplasts from one with separation of chromosomes a later, derived function. The telophase spindle separates the newly forming cytoplasts and the overlap between half spindles (the shared edge of two new domains) dictates the position at which cytokinesis occurs. Wall MTs of higher plant cells, like the MT cytoskeleton in animal and protistan cells, spatially define the interphase cytoplast. Redeployment of actin and MTs into the preprophase band (PPB) is the overt signal that the boundary between two nascent cytoplasts has been delineated. The "actin-depleted zone" that marks the site of the PPB throughout mitosis may be a more persistent manifestation of this delineation of two domains of cortical actin. The growth of the phragmoplast is controlled by these domains, not just by the spindle. These domains play a major role in controlling the path of phragmoplast expansion. Primitive land plants show different morphological changes that reveal that the plane of division, with or without the PPB, has been determined well in advance of mitosis.The green alga Spirogyra suggests how the phragmoplast system might have evolved: cytokinesis starts with cleavage and then actin-related determinants stimulate and positionally control cell-plate formation in a phragmoplast arising from interzonal MTs from the spindle. Actin in the PPB of higher plants may be assembling into a potential furrow, imprinting a cleavage site whose persistent determinants (perhaps actin) align the outgrowing edge of the phragmoplast, as in Spirogyra. Cytochalasin spatially disrupts polarized mitosis and positioning of the phragmoplast. Thus, the tensegral interaction of actin with MTs (at the spindle pole and in the phragmoplast) is critical to morphogenesis, just as they seem to be during division of animal cells. In advanced green plants, intercalary expansion driven by turgor is controlled by MTs, which in conjunction with actin, may act as stress detectors, thereby affecting the plane of division (a response clearly evident after wounding of tissue). The PPB might be one manifestation of this strain detection apparatus.     

F-actin-dependent endocytosis of cell wall pectins in meristematic root cells. Insights from brefeldin A-induced compartments

Brefeldin A (BFA) inhibits exocytosis but allows endocytosis, making it a valuable agent to identify molecules that recycle at cell peripheries. In plants, formation of large intracellular compartments in response to BFA treatment is a unique feature of some, but not all, cells. Here, we have analyzed assembly and distribution of BFA compartments in development- and tissue-specific contexts of growing maize (Zea mays) root apices. Surprisingly, these unique compartments formed only in meristematic cells of the root body. On the other hand, BFA compartments were absent from secretory cells of root cap periphery, metaxylem cells, and most elongating cells, all of which are active in exocytosis. We report that cell wall pectin epitopes counting rhamnogalacturonan II dimers cross-linked by borate diol diester, partially esterified (up to 40%) homogalacturonan pectins, and (1-->4)-beta-D-galactan side chains of rhamnogalacturonan I were internalized into BFA compartments. In contrast, Golgi-derived secretory (esterified up to 80%) homogalacturonan pectins localized to the cytoplasm in control cells and did not accumulate within characteristic BFA compartments. Latrunculin B-mediated depolymerization of F-actin inhibited internalization and accumulation of cell wall pectins within intracellular BFA compartments. Importantly, cold treatment and protoplasting prevented internalization of wall pectins into root cells upon BFA treatment. These observations suggest that cell wall pectins of meristematic maize root cells undergo rapid endocytosis in an F-actin-dependent manner.     

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

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

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.     

Involvement of the Golgi region in the intracellular trafficking of cholera toxin

The intracellular pathway following receptor-mediated endocytosis of cholera toxin was studied using brefeldin A (BFA), which inhibited protein secretion and induced dramatic morphological changes in the Golgi region. In both mouse Y1 adrenal cells and CHO cells, BFA at 1 μg/ml caused a 80–90% inhibition of the cholera toxin (CT)-elevation of intracellular cAMP. The inhibition of the cytotoxicity of CT by BFA was also observed in a rounding assay of Y1 adrenal cells. The inhibition of CT cytotoxicity by BFA was dose dependent, with the ID₅₀ value similar to the LD₅₀ of BFA in Y1 adrenal cells. Binding and internalization of [¹²⁵I]-cholera toxin in Y1 adrenal cells was not affected by BFA. Unlike the BFA-sensitive cell lines such as Y1 adrenal and CHO cells, BFA at 1 μg/ml did not inhibit the cytotoxicity of CT in PtK₁ cells, of which the Golgi structure was BFA-resistant. These results strongly suggest that a BFA-sensitive Golgi is required for the protection of CT cytotoxicity by BFA. In contrast, elevation of the intracellular cAMP by forskolin, which acts directly on the plasma membrane adenylate cyclase, was not affected by BFA. These observations indicate that the intoxication of target cells by CT requires an intact Golgi region for its intracellular trafficking and/or processing. In this respect, CT shares a common intracellular pathway with ricin, Pseudomonas toxin, and modeccin, even though their structures and modes of action are very different. © 1993 Wiley-Liss, Inc.     

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.     

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