Preprophase Band Research Articles

Cyclin/Cdk complexes: their involvement in cell cycle progression and mitotic division.

DNA replication and mitosis are dependent on the activity of cyclin-dependent protein kinase (CDK) enzymes, which are heterodimers of a catalytic subunit with a cyclin subunit. Cyclin binding to specific individual proteins is thought to provide potential substrates to Cdk. Protein binding by cyclins is assessed in terms of its mechanisms and biological significance, using evidence from diverse organisms including substrate specificity in animal Cdk enzymes containing D-, A-, and B-type cyclins and extensive cyclin gene manipulations in yeasts. Assembly of protein complexes with cyclin/Cdk is noted and the capacity of the cyclin-dependent kinase subunit Cks, in such complex, to extend the range of Cdk substrates is documented and discussed in terms of cell cycle regulation. Cell cycle progression involves changing abundance of individual cyclins, due to changing rates of their transcription or proteolysis, with consequent changes in the substrates of CDK through the cell cycle. Some overlap of the functions of individual cyclins in vivo has been identified by cyclin deletions and is suggested to follow a pattern in which cyclins can commonly complete functions initiated by the preceding cyclins well enough to preserve viability as groups of cyclins are removed by proteolysis. Cyclin accumulation is particularly important in terminating the G1 phase, when it raises CDK activity and starts events leading to DNA replication. It is suggested that plants share this mechanism. The distribution of cyclins and Cdk in maize root tip cells during mitosis and cytokinesis indicates the presence of Cdk1 (Cdc2a) and cyclin CycB1zm;2 at the mature and disassembling preprophase band and the presence of CycB1zm;2 at condensing and condensed chromosomes. Both observations correlate with the earlier-reported capacity of injected metaphase cyclin/CDK to accelerate preprophase band disassembly and chromosome condensation and with observations of the location of Cdk and cyclins in other laboratories. Additionally CycB1zm;2 is seen at the nuclear envelope during its breakdown, which correlates with an acceleration of the process by injected metaphase cyclin B/CDK. A phenomenon possibly unique to the plant kingdom is the persistence of mitotic cyclins after anaphase. Participation of cyclins in cytokinesis is indicated by the concentration of the mitotic cyclin CycA1;zm;1 at the phragmoplast. It is suggested that cyclins have a general function of spatially focusing Cdk activity and that in the plant cell the concentrations of cyclins are important mediators of CDK activity at the cytoskeleton, chromosomes, spindle, nuclear envelope, and phragmoplast.

CDNA isolation, characterization, and protein intracellular localization of a katanin-like p60 subunit from Arabidopsis thaliana.

Katanin, a heterodimeric protein with ATP-dependent microtubule-severing activity, localizes to the centrosome in animal cells. Widespread occurrence is suspected as several species contain homologs to the katanin p60 subunit. Recently we isolated an Arabidopsis thaliana cDNA with significant identity to the p60 subunit of sea urchin katanin. Like p60, the encoded protein is a member of the AAA superfamily of ATPases, containing the Walker ATP binding consensus and the signature AAA minimal consensus sequences within a single larger AAA/CAD amino acid motif. Phylogenetic analysis placed the encoded protein in the AAA subfamily of cytoskeleton-interactive proteins, where it formed a strongly supported clade with 4 other members identified as katanin p60 subunits. The clone was named AtKSS (Arabidopsis thaliana katanin-like protein small subunit). Western blots, performed using a polyclonal antibody raised against recombinant AtKSS, revealed AtKSS is present in protein extracts of all Arabidopsis organs examined. To evaluate potential interactions between AtKSS and the cytoskeleton, the intracellular localization of AtKSS was correlated with that of tubulin. AtKSS was found in perinuclear regions during interphase, surrounding the spindle poles during mitosis, but was absent from the preprophase band and phragmoplast microtubule arrays. These data support the thesis that AtKSS is an Arabidopsis homolog of the p60 subunit of katanin. Its cell cycle-dependent distribution is consistent with microtubule-severing activity, but additional studies will better define its role.

Endoplasmic reticulum preprophase band in dividing root-tip cells of Pinus brutia.

In dividing root-tip cells of Pinus brutia Ten., immunolocalization of the luminal endoplasmic reticulum (ER) proteins, which have the C-terminal HDEL sequence, reveals that the ER is reorganized during the preprophase/prophase stage. Portions of ER were arrayed into a ring-like structure at the site of the microtubule preprophase band (Mt-PPB). This preprophase ER band (ER-PPB) resembles that of the Mt-PPB. The former undergoes a maturation process closely similar to that of the latter. Our data show that the PPB region has a more complex organization than is currently believed. The probable function(s) of the ER-PPB is discussed.

Dynamic recruitment of Cdc2 to specific microtubule structures during mitosis.

A-type cyclin-dependent kinases (CDKs), also known as cdc2, are central to the orderly progression of the cell cycle. We made a functional Green Fluorescent Protein (GFP) fusion with CDK-A (Cdc2-GFP) and followed its subcellular localization during the cell cycle in tobacco cells. During interphase, the Cdc2-GFP fusion protein was found in both the cytoplasm and the nucleus, where it was highly resistant to extraction. In premitotic cells, a bright and narrow equatorial band appeared on the cell surface, resembling the late preprophase band, which disintegrated within 10 min as followed by time-lapse images. Cdc2-GFP was not found on prophase spindles but left the chromatin soon after this stage and associated progressively with the metaphase spindle in a microtubule-dependent manner. Arresting cells in mitosis through the stabilization of microtubules by taxol further enhanced the spindle-localized pool of Cdc2-GFP. Toward the end of mitosis, Cdc2-GFP was found at the midzone of the anaphase spindle and phragmoplast; eventually, it became focused at the midline of these microtubule structures. In detergent-extracted cells, the Cdc2-GFP remained associated with mitotic structures. Retention on spindles was prevented by pretreatment with the CDK-specific inhibitor roscovitine and was enhanced by the protein phosphatase inhibitor okadaic acid. Furthermore, we demonstrate that both the endogenous CDK-A and Cdc2-GFP were cosedimented with taxol-stabilized plant microtubules from cell extracts and that Cdc2 activity was detected together with a fraction of polymerized tubulin. These data provide evidence that the A-type CDKs associate physically with mitotic structures in a microtubule-dependent manner and may be involved in regulating the behavior of specific microtubule arrays throughout mitosis.

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.

A γ-tubulin antibody against a plant peptide sequence localises to cell division-specific microtubule arrays and organelles in plants

Gamma tubulin ( γ-tubulin) is involved in microtubule initiation in the eukaryotes. In animal cells it is localised to centrosomes and to other, non-centrosomal sites of microtubule initiation. In addition, cytoplasmic complexes containing γ-tubulin ( γ-TuRCs; γ-somes) have been described, which are multiprotein complexes involved in microtubule initiation. Most localisations of γ-tubulin in plants have previously been achieved using an antibody directed towards a conserved peptide sequence found in animal cells, showing co-localisation with all microtubule arrays throughout the cell cycle. Because different antibodies may give various patterns of subcellular localisation, in the present study we raised a polyclonal antibody (‘Hayley’) to the plant peptide sequence E DFAT QG GDRKDVFFY (bold letters indicate plant-specific amino acids) to further investigate the subcellular distribution in plants. Immunoblotting using wheat root tip protein extracts revealed a 58 kDa γ-tubulin-like peptide as has been described before. Immunofluorescence microscopy of wheat root-tip cells, however, revealed localisation of γ-tubulin to a subset of mitotic microtubule arrays and the cytokinetic phragmoplast, but not to interphase cortical arrays or the preprophase band of microtubules. This lack of labelling may be caused by a restriction of antibody access during interphase, but more likely by a cell division-specific conformational change in the γ-tubulin molecule. Our antibody also gave an organelle-like labelling, not described before, which may represent storage forms or precursors of γ-tubulin, perhaps related to plastid-based microtubule initiation in hepatics and hornworts.

Cambial reactivation in locally heated stems of the evergreen conifer Abies sachalinensis (Schmidt) Masters

A study was made of cambial activity, the localization of storage starch around the cambium, and the localization and occurrence of microtubules in cambial cells from dormancy to reactivation in locally heated (22-26 degrees C) stems of the evergreen conifer Abies sachalinensis. Heating induced localized reactivation of the cambium in the heated portions of the stem. Erect ray cambial cells resumed cell division 1 d prior to the reactivation of fusiform cambial cells and procumbent ray cambial cells. The re-initiation of the division of fusiform cambial cells occurred first on the phloem side. During the heat treatment, the amount of storage starch decreased in procumbent ray cambial cells and in the phloem parenchyma adjacent to the cambium but increased in fusiform cambial cells. Preprophase bands of microtubules, spindle microtubules and phragmoplast microtubules were observed both in erect ray cambial cells and in procumbent ray cambial cells. By contrast, no evidence of the presence of such preprophase bands of microtubules was detected in fusiform cambial cells. The results suggest that the localized heating of stems of evergreen conifers might provide a useful experimental model system for studies of the dynamics of cambial reactivation in intact trees.

The cytoskeleton and spatial control of cytokinesis in the plant life cycle

One of the intriguing aspects of development in plants is the precise control of division plane and subsequent placement of walls resulting in the specific architecture of tissues and organs. The placement of walls can be directed by either of two microtubule cycles. The better known microtubule cycle is associated with control of the future division plane in meristematic growth where new cells become part of tissues. The future daughter domains are determined before the nucleus enters prophase and the future site of cytokinesis is marked by a preprophase band (PPB) of cortical microtubules. The spindle axis is then organized in accordance with the PPB and, following chromosome movement, a phragmoplast is initiated in the interzone and expands to join with parental walls at the site previously occupied by the PPB. The alternative microtubule cycle lacks both the hooplike cortical microtubules of interphase and the PPB. Wall placement is determined by a radial microtubule system that defines a domain of cytoplasm either containing a nucleus or destined to contain a nucleus (the nuclear cytoplasmic domain) and controls wall placement at its perimeter. This more flexible system allows for cytoplasmic polarization and migration of nuclei in coenocytes prior to cellularization. The uncoupling of cytokinesis from karyokinesis is a regular feature of the reproductive phase in plants and results in specific, often unusual, patterns of cells which reflect the position of nuclei at the time of cellularization (e.g., the arrangement of spores in a tetrad, cells of the male and female gametophytes of angiosperms, and the distinctive cellularization of endosperm). Thus, both microtubule cycles are required for completion of plant life cycles from bryophytes to angiosperms. In angiosperm seed development, the two methods of determining the boundaries of domains where walls will be deposited are operative side by side. Whereas the PPB cycle drives embryo development, the radial-microtubule-system cycle drives the common nuclear type of endosperm development from the syncytial stage through cellularization. However, a switch to the PPB cycle can occur in endosperm, as it does in barley, when peripheral cells divide to produce a multilayered aleurone. The triggers for the switch between microtubule cycles, which are currently unknown, are key to understanding plant development.

Plasma membrane-cell wall connections: roles in mitosis and cytokinesis revealed by plasmolysis of Tradescantia virginiana leaf epidermal cells.

Tradescantia virginiana leaf epidermal cells were plasmolysed by sequential treatment with 0.8 M and 0.3 M sucrose. Plasmolysis revealed adhesion of the plasma membrane to the cell wall at sites coinciding with cytoskeletal arrays involved in the polarisation of cells undergoing asymmetric divisions--cortical actin patch--and in the establishment and maintenance of the division site--preprophase band of microtubules and filamentous (F) actin. The majority of cells retained adhesions at the actin patch throughout mitosis. However, only approximately 13% of cells formed or retained attachments at the site of the preprophase band. After the breakdown of the nuclear envelope, plasmolysis had a dramatic effect on spindle orientation, cell plate formation, and the plane of cytokinesis. Spindles were rotated at abnormal angles including tilted into the plane of the epidermis. Cell plates formed but were quickly replaced by vacuole-like intercellular compartments containing no Tinopal-stainable cell wall material. This compartment usually opened to the apoplast at one side, and cytokinesis was completed by the furrow extending across the protoplast. This atypical cytokinesis was facilitated by a phragmoplast containing microtubules and F-actin. Progression of the furrow was unaffected by 25 micrograms of cytochalasin B per ml but inhibited by 10 microM oryzalin. Phragmoplasts were contorted and misguided and cytokinesis prolonged, indicating severe disruption to the guidance mechanisms controlling phragmoplast expansion. These results are discussed in terms of cytoskeleton-plasma membrane-cell wall connections that could be important to the localisation of plasma membrane molecules defining the cortical division site and hence providing positional information to the cytokinetic apparatus, and/or for providing an anchor for cytoplasmic F-actin necessary to generate tension on the phragmoplast and facilitate its directed, planar expansion.

Use of abnormal preprophase bands to decipher division plane determination.

Many premitotic plant cells possess a cortical preprophase band of microtubules and actin filaments that encircles the nucleus. In vacuolated cells, the preprophase band is visibly connected to the nucleus by a cytoplasmic raft of actin filaments and microtubules termed the phragmosome. Typically, the location of the preprophase band and phragmosome corresponds to, and thus is thought to influence, the location of the cell division plane. To better understand the function of the preprophase band and phragmosome in orienting division, we used a green fluorescent protein-based microtubule reporter protein to observe mitosis in living tobacco bright yellow 2 cells possessing unusual preprophase bands. Observations of mitosis in these unusual cells support the involvement of the preprophase band/phragmosome in properly positioning the preprophase nucleus, influencing spindle orientation such that the cytokinetic phragmoplast initially grows in an appropriate direction, and delineating a region in the cell cortex that attracts microtubules and directs later stages of phragmoplast growth. Thus, the preprophase band/phragmosome appears to perform several interrelated functions to orient the division plane. However, functional information associated with the preprophase band is not always used or needed and there appears to be an age or distance-dependent character to the information. Cells treated with the anti-actin drug, latrunculin B, are still able to position the preprophase nucleus suggesting that microtubules may play a dominant role in premitotic positioning. Furthermore, in treated cells, spindle location and phragmoplast insertion are frequently abnormal suggesting that actin plays a significant role in nuclear anchoring and phragmoplast guidance. Thus, the microtubule and actin components of the preprophase band/phragmosome execute complementary activities to ensure proper orientation of the division plane.

Characterization of a mitotic mutant of durum wheat.

An ethyl methanesulfonate-induced mitotic mutant of durum wheat (Triticum turgidum L. var. durum; 2n = 4x = 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 (8x = 56, or 16x = 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.

Dynamic Organization of Microtubules and Microfilaments during Cell Cycle Progression in Higher Plant Cells

Abstract: The cytoskeleton, which mainly consists of microtubules (MTs) and actin microfilaments (MFs), plays various significant roles that are indispensable for eukaryotic viability, including determination of cell shape, cell movement, nuclear division, and cytokinesis. In animal cells, MFs appear to be of more importance than MTs, except for spindle formation in nuclear division. In contrast, higher plants have a rigid cell wall around their cells, and have thus evolved elegant systems of MTs to control the direction of cellulose microfibrils (CMFs) deposited in the cell wall, and to divide centrifugally in a physically limited space. Dynamic changes in MTs during cell cycle progression in higher plant cells have been observed over several decades, including cortical MTs (CMTs) during interphase, preprophase bands (PPBs) from late G2 phase to prophase, spindles from prometaphase to anaphase, and phragmoplasts at telophase. The MFs also show some changes not as obvious as MT dynamics. However, questions regarding the process of formation of these arrays, and the precise mechanisms by which they fulfill their roles, remain unsolved. In this article, we present an outline of the changes in the cytoskeleton based on our studies with highly‐synchronized tobacco BY‐2 cells. Some candidate molecules that could play roles in cytoskeletal dynamics are discussed. We also hope to draw attention to recent attempts at visualization of cytoskeletons with molecular techniques, and to some examples of genetic approaches in this field.

Two kinesin-related proteins associated with the cold-stable cytoskeleton of carrot cells: characterization of a novel kinesin, DcKRP120-2.

We have previously described the biochemical isolation of 65 kDa and 120 kDa microtubule-associated proteins from carrot cytoskeletons. The 65 kDa MAPs have subsequently been shown to be structural MAPs that reconstitute 30 nm cross-bridges of the kind that maintain cortical microtubules in parallel groups. By exploiting its avid binding to microtubules, we have now devised a method for isolating MAP120 from protoplast extracts, and shown that it has properties of a kinesin-related protein. MAP120 segregates with the cold stable pool of microtubules in carrot cytoskeletons, whilst the 65 kDa MAPs are also associated with the cold-sensitive microtubules. On gradient gels, MAP120 resolves as two kinesin-like bands. We report the isolation of a carrot cDNA, DcKRP120-2, corresponding to a novel kinesin of the BimC class known to move to the plus ends of microtubules. Antibodies raised against specific expressed sequences recognize the upper band, while the lower band is recognized by antibodies to the tobacco kinesin-related protein, TKRP125. We have also isolated a partial genomic carrot DNA, DcKRP120-1, homologous to the motor region of tobacco TKRP125. Immunofluorescence of the two proteins produces different staining patterns. Anti-TKRP125 labels the cortical microtubules and the pre-prophase band, but anti-DcKRP120-2 does so only weakly. Both clearly stain the spindle and the phragmoplast, but in a proportion of cells anti-DcKRP120-2 strongly decorates the phragmoplast mid-line where the plus ends of the microtubules overlap. We discuss the potential roles of these proteins during the microtubule cycle.

Rearrangement of the actin filament and microtubule cytoskeleton during induction of microspore embryogenesis inBrassica napus L. cv. Topas

Changes in the actin filament and microtubule cytoskeleton were examined during heat- and cytochalasin D-induced embryogenesis in microspores ofBrassica napus cv. Topas by rhodamine phalloidin and immunofluorescence labelling respectively. The nucleus was displaced from its peripheral to a more central position in the cell, and perinuclear actin microfilaments and microtubules extended onto the cytoplasm. Heat treatment induced the formation of a preprophase band of microtubules in microspores; preprophase bands are not associated with the first pollen mitosis. Actin filament association with the preprophase band was not observed. The orientation and position of the mitotic spindle were altered, and it was surrounded with randomly oriented microfilaments. The phragmoplast contained microfilaments and microtubules, as in pollen mitosis I, but it assumed a more central position. Cytoskeletal reorganisation also occurred in microspores subjected to a short cytochalasin D treatment, in the absence of a heat treatment. Cytochalasin D treatment of microspores resulted in dislocated mitotic spindles, disrupted phragmoplasts, and symmetric divisions and led to embryogenesis, confirming that a normal actin cytoskeleton has a role in preventing the induction of embryogenesis.

Salt stress induces changes in amounts and localization of the mitogen-activated protein kinase SIMK in alfalfa roots

SIMK is an alfalfa mitogen-activated protein kinase (MAPK) that is activated by salt stress and shows a nuclear localization in suspension-cultured cells. We investigated the localization of SIMK in alfalfa (Medicago sati a) roots. Although SIMK was expressed in most tissues of the root apex, cells of the quiescent center and statocytes showed much lower SIMK protein amounts. In cells of the elongation zone, SIMK was present in much higher amounts in epidermal than in cortex cells. In dividing cells of the root tip, SIMK revealed a cell cycle phase-dependent localization, being predominantly nuclear in interphase but associating with the cell plate and the newly formed cell wall in telophase and early G1 phase. In dividing cells, salt stress resulted in an association of part of the SIMK with the preprophase band. Generally, salt stress resulted in much higher amounts of SIMK in dividing cells of the root apex and epidermal cells of the elongation zone. These data demonstrate that amounts and subcellular localization of SIMK in roots is highly regulated and sensitive to environmental stress.

Gamma-tubulin colocalizes with microtubule arrays and tubulin paracrystals in dividing vegetative cells of higher plants

The distribution of γ-tubulin throughout cell division is studied in several taxa of higher plants. γ-Tubulin is present along the whole length of microtubules (Mts) in every cell stage-specific Mt array such as the preprophase band, the preprophase-prophase perinuclear Mts, the kinetochore Mt bundles, the phragmoplast, and the telophase-interphase transition Mt arrays. γ-Tubulin follows with precision the Mt pattern, being absent from any other, Mt-free, cell site. In cells treated with anti-Mt drugs, γ-tubulin is present only on degrading or on reappearing Mt arrays, while it is totally absent from cells devoid of Mts. γ-Tubulin is also present in tubulin paracrystals, which are formed in colchicine-treated cells. These observations support the view that in higher plants γ-tubulin may not be a microtubule-organizing-center-specific protein, but it may play a certain structural and/or functional role being related to α- and β-tubulin.

Plant chaperonins: a role in microtubule-dependent wall formation?

The cytosolic chaperonin containing t-complex peptide-1 (CCT) is involved in the correct folding of newly synthetized actin and tubulin molecules. To get insight into potential additional functions of plant CCT, the localization of the subunit CCTɛ was followed throughout cell cycle, cell elongation, and cell differentiation in the tobacco cell culture VBI-O with relation to the microtubular cytoskeleton by double-immunofluorescence and confocal microscopy. The CCTɛ subunit was found to colocalize with sites of microtubule nucleation such as nuclear envelope and preprophase band. In addition, CCTɛ was associated with tubulin in sites of elevated wall synthesis such as phragmoplast or along secondary-wall thickenings. CCTɛ and its substrate tubulin were found to be soluble during periods of cytoskeletal dynamics, whereas sedimentable, vesicle-bound forms of CCTɛ and tubulin prevailed during cell differentiation. The sedimentability of CCTɛ was increased by calcium, whereas it was detached from microsomes by ATP. CCTɛ can bind to both polymerized microtubules and tubulin dimers. These data suggest an additional function of plant CCT in microtubule-driven transport of vesicles that contain cell-wall material.

Intracellular distribution of subcellular organelles revealed by antibody against xyloglucan during cell cycle in tobacco BY-2 cells

Immunofluorescence microscopy using an antibody against xyloglucan (XG) revealed its dynamics during the cell cycle. In interphase tobacco BY-2 cells, punctate and scattered fluorescence was observed throughout the cytoplasm. Colocalization of such signals with cortical microtubules (MTs) was clearly observed on the membrane ghosts. They were also associated and accumulated on MT bundles of the preprophase band. Treatment of protoplasts with cytochalasin B prior to the preparation of the ghosts had no effect on the pattern of anti-XG staining, while treatment with propyzamide caused the disappearance of the staining. These results suggest an association of Golgi apparatus and/or Golgi-derived vesicles with MTs. In metaphase cells, the staining was dispersed in the cytoplasm, except in the area occupied by the metaphase spindle. During anaphase, a broad fluorescence band appeared between daughter chromosomes and gradually concentrated at the equatorial plane before formation of the phragmoplast. At telophase, a bright line of fluorescence appeared at the equatorial plane corresponding to the position of the cell plate. The length of the line increased as cytokinesis proceeded. Thus, we showed that immunofluorescence microscopy using anti-XG antibody can be considered as a powerful tool for the analysis of Golgi apparatus and Golgi-derived vesicles containing XG.

Redistribution of Golgi stacks and other organelles during mitosis and cytokinesis in plant cells.

We have followed the redistribution of Golgi stacks during mitosis and cytokinesis in living tobacco BY-2 suspension culture cells by means of a green fluorescent protein-tagged soybean alpha-1,2 mannosidase, and correlated the findings to cytoskeletal rearrangements and to the redistribution of endoplasmic reticulum, mitochondria, and plastids. In preparation for cell division, when the general streaming of Golgi stacks stops, about one-third of the peripheral Golgi stacks redistributes to the perinuclear cytoplasm, the phragmosome, thereby reversing the ratio of interior to cortical Golgi from 2:3 to 3:2. During metaphase, approximately 20% of all Golgi stacks aggregate in the immediate vicinity of the mitotic spindle and a similar number becomes concentrated in an equatorial region under the plasma membrane. This latter localization, the "Golgi belt," accurately predicts the future site of cell division, and thus forms a novel marker for this region after the disassembly of the preprophase band. During telophase and cytokinesis, many Golgi stacks redistribute around the phragmoplast where the cell plate is formed. At the end of cytokinesis, the daughter cells have very similar Golgi stack densities. The sites of preferential Golgi stack localization are specific for this organelle and largely exclude mitochondria and plastids, although some mitochondria can approach the phragmoplast. This segregation of organelles is first observed in metaphase and persists until completion of cytokinesis. Maintenance of the distinct localizations does not depend on intact actin filaments or microtubules, although the mitotic spindle appears to play a major role in organizing the organelle distribution patterns. The redistribution of Golgi stacks during mitosis and cytokinesis is consistent with the hypothesis that Golgi stacks are repositioned to ensure equal partitioning between daughter cells as well as rapid cell plate assembly.

Inhibition of serine/threonine-specific protein phosphatases causes premature activation of cdc2MsF kinase at G2/M transition and early mitotic microtubule organisation in alfalfa.

Reversible phosphorylation of serine/threonine residues of cell cycle-regulatory proteins is one of the key molecular mechanisms controlling eukaryotic cell division. In plants, the protein kinase partners (i.e. p34cdc2/CDC28-related kinases) have been extensively studied, while the role of counter-acting protein phosphatases is less well understood. We used endothall (ET) as a cell-permeable inhibitor of serine/threonine-specific protein phosphatases to alter cytological and biochemical characteristics of cell division in cultured alfalfa cells. A high concentration of ET (10 and 50 microM) inhibited both protein phosphatases 1 and 2 (PP1 and PP2A), while a low concentration (1 microM) of ET-treatment primarily reduced the PP2A activity. High concentrations of the inhibitor increased the frequency of hypercondensed early and late prophase chromosomes that could not enter metaphase. In contrast, a low concentration of ET did not interfere with chromosomal events but caused significant alterations in the organisation of microtubules. Exposure of cells to 1 microM ET resulted in disturbance of preprophase band formation, increase in the number of nuclei with prophase microtubule assembly, premature polarisation of the spindle, and abnormal phragmoplast maturation. Under the same conditions, the ET-treated cells exhibited an early increase in cdc2MsF kinase activity. These results suggest that PP2A contributes to the control of mitotic kinase activities and microtubule organisation. Normal chromosome condensation and mitotic progression are dependent on both PP1 and PP2A activities. The presented data support the functional role of protein phosphatases in the co-ordination of chromosomal and microtubule events in dividing plant cells.