Spatio-temporal relationship between nuclear-envelope breakdown and preprophase band disappearance in cultured tobacco cells

Cell division involves the coordinated progression of karyokinesis and cytokinesis, which is accomplished by communication between the nucleus and the cytoplasm. We have utilized green-fluorescent-protein technology to generate a line of tobacco 'Bright Yellow 2' (BY-2) cells labeled for both microtubules and the nuclear envelope. This cell line allowed us to use living cells to investigate the relationship between nuclear-envelope breakdown and preprophase band disappearance with high spatial and temporal resolution. Our observations demonstrate that nuclear-envelope breakdown always precedes preprophase band disappearance in BY-2 cells. In addition, the rate of preprophase band disappearance, and the attenuation of perinuclear microtubule fluorescence, correlates with the proximity of the nucleus to the preprophase band site. These results indicate the presence of communication between the nucleus and the preprophase band and suggest a causal relationship between nuclear-envelope breakdown and preprophase band disappearance.     

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

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

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.     

The electronmicroscopical study of the division of highly vacuolised grapevine callus cells

The ultrastructural aspects of the cell division in the grapevine(Vitis riparia × V.labrusca) calli were studied. A large central vacuole plays a noticeable part in this process. Before its division the nucleus with some encircling cytosol moves into the central vacuole where the small, round-shaped portion of cytosol (phragmosome) originates. In this central mass of cytosol connected with the peripheral one by thin cytosolic strands karyokinesis is carried out and the cell plate formation starts. Before karyokinesis the phragmosome, however, does not exhibit the form of the cytosolic layer completely traversing the cell. No preprophase band of microtubules has been observed in the cells either. The polarity of the mitotic spindle designating the orientation of the new cell wall is random then and it is not determined by the position of the preprophase band of microtubules or by the orientation of phragmosome. The unorganized growth of the grapevine callus reflects this fact.     

discordia mutations specifically misorient asymmetric cell divisions during development of the maize leaf epidermis.

In plant cells, cytokinesis depends on a cytoskeletal structure called a phragmoplast, which directs the formation of a new cell wall between daughter nuclei after mitosis. The orientation of cell division depends on guidance of the phragmoplast during cytokinesis to a cortical site marked throughout prophase by another cytoskeletal structure called a preprophase band. Asymmetrically dividing cells become polarized and form asymmetric preprophase bands prior to mitosis; phragmoplasts are subsequently guided to these asymmetric cortical sites to form daughter cells of different shapes and/or sizes. Here we describe two new recessive mutations, discordia1 (dcd1) and discordia2 (dcd2), which disrupt the spatial regulation of cytokinesis during asymmetric cell divisions. Both mutations disrupt four classes of asymmetric cell divisions during the development of the maize leaf epidermis, without affecting the symmetric divisions through which most epidermal cells arise. The effects of dcd mutations on asymmetric cell division can be mimicked by cytochalasin D treatment, and divisions affected by dcd1 are hypersensitive to the effects of cytochalasin D. Analysis of actin and microtubule organization in these mutants showed no effect of either mutation on cell polarity, or on formation and localization of preprophase bands and spindles. In mutant cells, phragmoplasts in asymmetrically dividing cells are structurally normal and are initiated in the correct location, but often fail to move to the position formerly occupied by the preprophase band. We propose that dcd mutations disrupt an actin-dependent process necessary for the guidance of phragmoplasts during cytokinesis in asymmetrically dividing cells.     

Monoplastidic mitosis in the mossTimmiella barbuloides (Bryophyta)

Summary Mitotic cell division of monoplastidic sporogones was investigated in the mossTimmiella barbuloides (Brid.) Moenk. (Pottiales, Bryophyta) by TEM. Division polarity of sporogones is established by the interphase position of the single oblong cup-shaped plastid, which is orientated with its long axis parallel to one of the cell walls. In preprophase the plastid elongates and its extremities bend at right angles. Plastid growth is directed by microtubules and accompanied by plastid tubules. The plastid begins the process of duplication by constricting centrally in the plane of the future cytokinetic septum. There is no preprophase band of microtubules at the division site. The large central nucleus becomes fusiform and aligned parallel to the main plastid axis. By the end of prophase the daughter plastids are positioned at the opposite poles of the nucleus where they probably function as nucleating or organizing centres for the spindle microtubules. Metaphase and anaphase spindles contain long sheets of ER. Cytokinesis involves the formation of a well developed phragmoplast.Abbreviations TEM transmission electron microscopy - PPB preprophase band of microtubules - ER endoplasmic reticulum     

Preprophase band formation and cortical division zone establishment: RanGAP behaves differently from microtubules during their band formation

Correct positioning of the division plane is a prerequisite for plant morphogenesis. The preprophase band (PPB) is a key intracellular structure of division site determination. PPB forms in G2 phase as a broad band of microtubules (MTs) that narrows in prophase and specializes few-micrometer-wide cortical belt region, named the cortical division zone (CDZ), in late prophase. The PPB comprises several molecules, some of which act as MT band organization and others remain in the CDZ marking the correct insertion of the cell plate in telophase. Ran GTPase-activating protein (RanGAP) is accumulated in the CDZ and forms a RanGAP band in prophase. However, little is known about when and how RanGAPs gather in the CDZ, and especially with regard to their relationships to MT band formation. Here, we examined the spatial and temporal distribution of RanGAPs and MTs in the preprophase of onion root tip cells using confocal laser scanning microscopy and showed that the RanGAP band appeared in mid-prophase as the width of MT band was reduced to nearly 7 µm. Treatments with cytoskeletal inhibitors for 15 min caused thinning or broadening of the MT band but had little effects on RanGAP band in mid-prophase and most of late prophase cells. Detailed image analyses of the spatial distribution of RanGAP band and MT band showed that the RanGAP band positioned slightly beneath the MT band in mid-prophase. These results raise a possibility that RanGAP behaves differently from MTs during their band formation.     

Plastid apportionment and preprophase microtubule bands in monoplastidic root meristem cells ofIsoetes andSelaginella

Summary Ultrastructural observations on monoplastidic root tip cells ofIsoetes andSelaginella demonstrate two important phenomena associated with preprophasic preparation for mitotic cell division, 1. the preprophase band and 2. precise orientation of the dividing plastid relative to the preprophase band. Both of these phenomena accurately predict the future plane of cell division. The plastid divides in a plane parallel to the spindle and each cell inherits a single plastid which caps the telophase nucleus. When succesive transverse divisions occur, the plastid migrates prior to prophase from a position near an old transverse wall to a lateral position in the cell. The plastid is oriented with its median constriction precisely intersected by the plane of the preprophase band. When a longitudinal division follows a transverse division, the plastid remains in its position adjacent to an old transverse wall where it is bisected by the plane of the longitudinally oriented preprophase band microtubules.     

Cortical division zone establishment in plant cells

Plant cell division is spatially organized to maintain a critical cell volume and to control growth directionality. The correct orientation of the separating cell wall is secured by means of specialized cytoskeletal structures that guide the newly formed cell plate toward a predefined cortical position. A ring of microtubules called preprophase band defines a cortical zone that corresponds to the future division plane. Coincident with the disappearance of the preprophase band microtubules, cortical actin is removed at the corresponding position, leaving an actin-depleted zone that persists throughout mitosis. Here, we review the spatial and structural organization of the cortical division zone and discuss evidence that implicate the plasma membrane in division plane establishment.     

Roles of actin-depleted zone and preprophase band in determining the division site of higher-plant cells, a tobacco BY-2 cell line expressing GFP-tubulin

Summary. The mode of cytokinesis, especially in determining the site of cell division, is not well understood in higher-plant cells. The division site appears to be predicted by the preprophase band of microtubules that develop with the phragmosome, an intracellular structure of the cytoplasm suspending the nucleus and the mitotic apparatus in the center. As the preprophase band disappears during mitosis, it is thought to leave some form of memory on the plasma membrane to guide the growth of the new cell plate at cytokinesis. However, the intrinsic nature of this memory remains to be clarified. In addition to microtubules, microfilaments also dynamically change forms during cell cycle transition from the late G2 to the early G1 phase. We have studied the relationships between microtubules and microfilaments in tobacco BY-2 cells and transgenic BY-2 cells expressing a fusion protein of green-fluorescent protein and tubulin. At the late G2 phase, microfilaments colocalize with the preprophase band of microtubules. However, an actin-depleted zone which appears at late prometaphase is observed around the chromosomes, especially at metaphase, but also throughout anaphase. To study the functions of the actin-depleted zone, we disrupted the microfilament structures with bistheonellide A, a novel macrolide that depolymerizes microfilaments very rapidly even at low concentrations. The division planes became disorganized when the drug was added to synchronized BY-2 cells before the appearance of the actin-depleted zone. In contrast, the division planes appeared smooth, as in control cells, when the drug was added after the appearance of the actin-depleted zone. These results suggest that the actin-depleted zone may participate in the demarcation of the division site at the final stage of cell division in higher plants.Correspondence and reprints: Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwanoha 5-1-5, Kashiwa, Chiba Prefecture 277-8562, Japan.     

Microtubule reorganization accompanying preprophase band formation in guard mother cells ofAvena sativa L.

Summary In order to study developmental changes in microtubule organization attending the formation of a longitudinally oriented preprophase band, the guard mother cells ofAvena were examined using a new procedure for anti-tubulin immunocytochemistry on large epidermal segments. We found that the interphase band (IMB) of transverse cortical microtubules present in these cells following asymmetric division is replaced after subsidiary cell formation by mesh-like to radial microtubules that extend throughout the cytoplasm. Many of the Mts are also grouped in bundles. Gradually, this intermediate array is succeeded by longitudinal elements of the PPB. Thus, preprophase band formation is accompanied by a 90° shift in Mt orientation, with a radial arrangement serving as an intermediate stage. The micrographs are most consistent with the rearrangement of intact Mts, although changes in Mt assembly are possible as well. The role of the IMB in guard mother cells is also discussed.Abbreviations GMC guard mother cell - IMB interphase microtubule band - Mt microtubule - PPB preprophase band     

Microtubule-associated AIR9 recognizes the cortical division site at preprophase and cell-plate insertion

In plants, the preprophase band (PPB) of microtubules marks the cortical site where the cross-wall will fuse with the parental wall during cytokinesis . This band disappears before metaphase, and it is not known how the division plane is "memorized". One idea is that the PPB leaves behind molecules involved in the maturation of the cell plate . Here, we report on the proteomic isolation of a novel 187 kDa microtubule-associated protein, AIR9, conserved in land plants and trypanosomatid parasites. AIR9 decorates cortical microtubules and the PPB but is downregulated during mitosis. AIR9 reappears at the former PPB site precisely when the cortex is contacted by the outwardly growing cytokinetic apparatus. AIR9 then moves inward on the new cross-wall and thus forms a torus. Truncation studies show that formation of the torus requires a repeated domain separate from AIR9's microtubule binding site. Cell plates induced to insert outside the predicted division site do not elicit an AIR9 torus, suggesting that AIR9 recognizes a component of the former PPB. Such misplaced walls remain immature, based on their prolonged staining for the cell-plate polymer callose. We propose that AIR9 may be part of the mechanism ensuring the maturation of those cell plates successfully contacting the "programmed" cortical division site.     

Preprophasic establishment of division polarity in monoplastidic mitosis of hornworts

Summary Preprophase in the monoplastidic mitotic cells ofPhaeoceros andNotothylas is characterized by the establishment of a division site in the absence of a typical preprophase band. The future cytokinetic plane is predicted by plastid orientation and development of an elaborate preprophasic microtubule system perpendicular to the division plane. Division of the single plastid is initiated early in preprophase and the constricting plastid migrates to a position perpendicular to the future plane of division. Plastid orientation assures that division of the plastid by mid-constriction will result in distribution of a plastid to each daughter cell. Microtubules parallel the long axis of the plastid and are most numerous adjacent to the nucleus which becomes elongated in the future spindle axis. We conclude that the division site is a fundamental component of the cytokinetic apparatus involved in the determination of cleavage plane prior to nuclear division.     

Plasma membrane-cell wall connections: Roles in mitosis and cytokinesis revealed by plasmolysis ofTradescantia virginiana leaf epidermal cells

Summary 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 g of cytochalasin B per ml but inhibited by 10 M 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.Abbreviations ADZ actin-depleted zone - DIC differential interference contrast - GMC guard mother cell - MT microtubule - PPB preprophase band - SMC subsidiary mother cell Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Demarcation of the cortical division zone in dividing plant cells

Somatic cytokinesis in higher plants involves, besides the actual construction of a new cell wall, also the determination of a division zone. Several proteins have been shown to play a part in the mechanism that somatic plant cells use to control the positioning of the new cell wall. Plant cells determine the division zone at an early stage of cell division and use a transient microtubular structure, the preprophase band (PPB), during this process. The PPB is formed at the division zone, leaving behind a mark that during cytokinesis is utilized by the phragmoplast to guide the expanding cell plate toward the correct cortical insertion site. This review discusses old and new observations with regard to mechanisms implicated in the orientation of cell division and determination of a cortical division zone.     

Monoclonal antibodies to a fern spermatozoid detect novel components of the mitotic and cytokinetic apparatus in higher plant cells

Summary The aim of this study was to search for uncharacterized components of the plant cytoskeleton using monoclonal antibodies raised against spermatozoids of the fernPteridium (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 thePteridium 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.Abbreviations MT microtubule - PPB preprophase band     

Regulation of the plane of cell division in vacuolated cells

Summary In small leaf explants fromNautilocalyx lynchii (Hook. f.) Sprague (Gesneriaceae) the vacuolated epidermal cells divide after 3–4 days. Most cells divide periclinally, but longitudinal and transverse divisions are also found. Before mitosis the cells form a phragmosome (PS), a cytoplasmic structure which contacts the cell cortex at the future division site. An experimental approach was used to find out at which time the plane of cell division becomes fixed: prior to or during the formation of a PS.When 3 day-old explants were divided into two parts by a longitudinal cut, a high percentage of the cells near the wound divided longitudinally. Cells which already had a PS at the time of wounding most often divided in the plane of the PS. Some of the cells with a non-longitudinal PS, however, formed a longitudinal cell wall after the replacement of the original PS by a longitudinal PS.The observations show that most cells which had not yet formed a PS could be induced to form a cell wall in a new direction. As soon as the formation of the PS had started, however, it became more difficult to induce a change in the plane of cell division. These results suggest that the division site is chosen during the formation of the PS.Abbreviations BMT band of microtubules - DIC differential interference contrast microscopy - l longitudinal - l-o longitudinal-oblique - MT microtubule - p periclinal - PM prometaphase - PPB preprophase band - PS phragmosome - t transverse - t-o transverse-oblique     

Alteration of microtubule dynamic instability during preprophase band formation revealed by yellow fluorescent protein-CLIP170 microtubule plus-end labeling

At the onset of mitosis, plant cells form a microtubular preprophase band that defines the plane of cell division, but the mechanism of its formation remains a mystery. Here, we describe the use of mammalian yellow fluorescent protein-tagged CLIP170 to visualize the dynamic plus ends of plant microtubules in transfected cowpea protoplasts and in stably transformed and dividing tobacco Bright Yellow 2 cells. Using plus-end labeling, we observed dynamic instability in different microtubular conformations in live plant cells. The interphase plant microtubules grow at 5 micro m/min, shrink at 20 micro m/min, and display catastrophe and rescue frequencies of 0.02 and 0.08 events/s, respectively, exhibiting faster turnover than their mammalian counterparts. Strikingly, during preprophase band formation, the growth rate and catastrophe frequency of plant microtubules double, whereas the shrinkage rate and rescue frequency remain unchanged, making microtubules shorter and more dynamic. Using these novel insights and four-dimensional time-lapse imaging data, we propose a model that can explain the mechanism by which changes in microtubule dynamic instability drive the dramatic rearrangements of microtubules during preprophase band and spindle formation in plant cells.     

discordia1 and alternative discordia1 Function Redundantly at the Cortical Division Site to Promote Preprophase Band Formation and Orient Division Planes in Maize

In plants, cell wall placement during cytokinesis is determined by the position of the preprophase band (PPB) and the subsequent expansion of the phragmoplast, which deposits the new cell wall, to the cortical division site delineated by the PPB. New cell walls are often incorrectly oriented during asymmetric cell divisions in the leaf epidermis of maize (Zea mays) discordia1 (dcd1) mutants, and this defect is associated with aberrant PPB formation in asymmetrically dividing cells. dcd1 was cloned and encodes a putative B' regulatory subunit of the PP2A phosphatase complex highly similar to Arabidopsis thaliana FASS/TONNEAU2, which is required for PPB formation. We also identified alternative discordia1 (add1), a second gene in maize nearly identical to dcd1. While loss of add1 function does not produce a noticeable phenotype, knock down of both genes in add1(RNAi) dcd1(RNAi) plants prevents PPB formation and causes misorientation of symmetric and asymmetric cell divisions. Immunolocalization studies with an antibody that recognizes both DCD1 and ADD1 showed that these proteins colocalize with PPBs and remain at the cortical division site through metaphase. Our results indicate that DCD1 and ADD1 function in PPB formation, that this function is more critical in asymmetrically dividing cells than in symmetrically dividing cells, and that DCD1/ADD1 may have other roles in addition to promoting PPB formation at the cortical division site.