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.     

FINE STRUCTURE STUDIES OF PHLEUM ROOT MERISTEM CELLS. II. MITOTIC ASYMMETRY AND CELLULAR DIFFERENTIATION

Avers , Charlotte J. (Douglass Coll., Rutgers—The State U., New Brunswick, N.J.) Fine structure studies of Phleum root meristem cells. II. Mitotic asymmetry and cellular differentiation. Amer. Jour. Bot. 50(2): 140–148. Illus. 1963.—An electron microscopical study showed that ultrastructural differences distinguished the cell dividing symmetrically from that undergoing asymmetrical division in timothy grass epidermis. The spindle orientation led to cytokinesis which produced either equal- or unequal-sized sister cells, but the mitotic apparatus itself varied in the mitoses. In asymmetrical cells, the basal pole showed more extensive endoplasmic reticulum (ER) polarizations, which intruded into the spindle area during metaphase and anaphase. Such ER polarity was not obvious in symmetrical mitosis or in the apical end of asymmetrically dividing cells. The mitotic sequence is described photographically. Foci of ER were observed as early as prophase in the polar region, and it is suggested that there is a resemblance to astral ray foci seen in prophase of animal cell mitosis. Cell plate formation could be detected in anaphase by accumulations of vesicles and ER fragments along the spindle equator. Phragmosomes apparently were not involved in cell plate formation in Phleum, unlike Allium, cytokinesis. The mitotic asymmetry is discussed as a consequence of an intracellular gradient separate from the intercellular gradient of differentiation along the entire length of growing root tip epidermis.     

Electron-microscope observations of mitosis and cytokinesis in multinucleate protoplasts of soybean.

Multinucleate soybean protoplasts produced by spontaneous fusion during enzyme digestion of the cell wall initiated cell division after approximately 40 h in culture. The structure of these protoplasts during mitosis and cytokinesis was studied with both light and electron microscopes. Most nuclei did not fuse but divided synchronously. Interphase nuclei was commonly connected by short narrow nuclear bridges. At prophase and metaphase the nuclei appeared typical of those in most higher plants; technical difficulties prevented an adequate examination of protoplasts at anaphase. Telophase was characterized by cytokinesis involving phragmoplast and cell plate formation; however, complete partitioning of the cytoplasm by cell plants was not observed. Numerous coated vesicles were present near to or continuous with the cell plate and plasmalemma. The presence of a few dividing protoplasts with at least double the normal chromosome number suggests that some nuclear fusion occurred prior to mitosis. Very little cell wall material was detected at the margin of the dividing protoplasts.     

Genetic dissection of cytokinesis

Higher plants have evolved specific mechanisms for partitioning the cytoplasm of dividing cells. In the predominant mode of phragmoplast-assisted cytokinesis, a cell wall and flanking plasma membranes are made de novo from a transient membrane compartment, the cell plate, which in turn forms by vesicle fusion from the centre to the periphery of the dividing cell. Other modes of cytokinesis appear to occur in meiotic cells and developing gametophytes. Here we review recent progress in the analysis of plant cytokinesis, focusing on genetic studies in Arabidopsis which are beginning to identify structural and regulatory components of phragmoplast-assisted cytokinesis. Two classes of mutations have been described. In one class, the defects appear to be confined to cell plate formation, suggesting that the execution of cytokinesis is specifically affected. Mutations in the other class display more general defects in cell division. We also discuss possible roles of proteins that have been localised in cytokinetic cells but not characterised genetically. Finally, mutations affecting meiotic or gametophytic cell divisions suggest that mechanistically different modes of cytokinesis occur in higher plants.     

Ultrastructure of vascular cambial cell cytokinesis in pine seedlings preserved by cryofixation and substitution

Summary.  Trees depend on the secondary vascular cambium to produce cells for new xylem and phloem. The fusiform cells of this lateral meristem are long and narrow, presenting special challenges for arranging the mitotic spindle and phragmoplast. Fusiform cambial cells of Pinus ponderosa and Pinus contorta were studied by cryofixation and cryosubstitution which preserved ultrastructure and phases of cytokinesis with a resolution not previously attained. Membranous structures including the plasma membrane, tonoplast, and those of other organelles were smooth and unbroken, indicating that they were preserved while the protoplasm was in a fully turgid state. Mitotic spindles separated daughter chromosomes diagonally across the radial width of the cells. The cell plate was initiated at an angle to the cell axis between the anaphase chromosomes by a microtubule array which organized vesicles at the phragmoplast midline. Within the phragmoplast, vesicles initially joined across thin tubular projections and then amalgamated into a tubulo-vesicular network. Axial expansion of the cell plate generated two opposing phragmoplasts connected by a thin, extended bridge of cell plate and cytoplasm that was oriented along the cell axis. In the cytoplasmic bridge trailing each phragmoplast, the callose-rich tubular network gradually consolidated into a fenestrated plate and then a complete cell wall. Where new membrane merged with old, the parent plasmalemma appeared to be loosened from the cell wall and the membranes joined via a short tubulo-vesicular network. These results have not been previously reported in cambial tissue, but the same phases of cytokinesis have been observed in cryofixed root tips and suspension-cultured cells of tobacco. Received February 11, 2002; accepted May 31, 2002; published online October 31, 2002 RID="*" ID="*" Correspondence and reprints: Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada. Abbreviations: CFS cryofixation and cryosubstitution; ER endoplasmic reticulum; HPF high-pressure freezing; PPB preprophase band.     

Origin of sperm cell association in the “male germ unit” ofBrassica pollen

The association of the two sperm cells inBrassica napus pollen following the generative cell division was investigated. The generative cell during division is located in the center of the pollen grain, within the vegetative cell. The space present between the two cells is slightly irregular as seen following standard glutaraldehyde fixation. After completion of mitosis vesicles appear in the equatorial plane, coalescing centripetally to form a cell plate which fuses with the membrane of the generative cell, dividing it in two sperm cells. They are isolated from the vegetative cell by the space between the two cell membranes and are separated from each other by a similar space resulting from the cell plate formed during cytokinesis.     

Simultaneous visualization of the actin plate and new cell partition membrane during cytokinesis in the brown alga Sphacelaria rigidula (Sphacelariales,Phaeophyceae)

In many brown algae, cytokinesis is accomplished through the centrifugal expansion of the membrane structure formed by the fusion of Golgi vesicles and flat cisternae. In contrast, it has been reported that cytokinesis in Sphacelaria rigidula progresses centripetally by adding Golgi vesicles and flat cisternae to cleaving furrows of the plasma membrane. The reason why this cytokinetic pattern was observed only in Sphacelaria species is unknown. In either cytokinesis pattern, a plate-like actin structure (the actin plate) coincides with the cytokinetic plane between the daughter nuclei. However, it is unclear how the actin plate is related to cytokinesis progression. In this study, we re-examined cytokinesis in the apical cells of S. rigidula using transmission electron microscopy. Double staining of the actin plate and the developing membrane was followed by fluorescence microscopy analysis to determine the relationship between these two formations. The results showed that cytokinesis in S. rigidula, as in many brown algae, was completed by centrifugal growth of the new cell partition membrane. A furrow of the plasma membrane was observed at the beginning of cytokinesis; however, further invagination did not occur. The actin plate arose at the center of the cytokinetic plane before membrane fusion and extended parallel to the expansion of the new cell partition membrane. When cytokinesis was slow due to insufficient Golgi vesicle supply to the cytokinetic plane in the cells under brefeldin A treatment, the extension of the actin plate was also suspended. In this study, the spatiotemporal relationship between the occurrence and expansion of the actin plate and the new cell partition membrane was revealed. These observations indicate that the actin plate might promote membrane fusion or lead to the growth of a new cell partition membrane.     

Cell division in leaves ofNicotiana

Summary Young leaves ofNicotiana tabacum were fixed in glutaraldehyde-formaldehyde followed by osmium tetroxide. The fine structure of dividing cells was studied. Before prophase a band of microtubules was observed between the nucleus and the cell wall at a position judged as the future plane of division. The microtubules in the band are 4–6 units deep and relatively closely packed, giving sections of the band a characteristic appearance. Micro-tubules of the mitotic spindle, the phragmoplast, and the preprophase band are morphologically similar. Some of the microtubules of the mitotic spindle and the phragmoplast have an undulate appearance. It is suggested that the undulate microtubules may have been fixed at a time when microwaves were traveling along them. The cell plate is formed by a fusion of small smooth surfaced vesicles and small coated vesicles. Fusion of small vesicles results first in larger vesicles and then in a meshwork of new cell-wall material surrounded by new regions of plasma membrane. Most of the vesicles are derived from dictyosomes and may be produced before and during prophase as well as during later stages of division. The ER may also contribute some vesicles to the cell plate.     

The Arabidopsis Exocyst Complex Is Involved in Cytokinesis and Cell Plate Maturation

Cell reproduction is a complex process involving whole cell structures and machineries in space and time, resulting in regulated distribution of endomembranes, organelles, and genomes between daughter cells. Secretory pathways supported by the activity of the Golgi apparatus play a crucial role in cytokinesis in plants. From the onset of phragmoplast initiation to the maturation of the cell plate, delivery of secretory vesicles is necessary to sustain successful daughter cell separation. Tethering of secretory vesicles at the plasma membrane is mediated by the evolutionarily conserved octameric exocyst complex. Using proteomic and cytologic approaches, we show that EXO84b is a subunit of the plant exocyst. Arabidopsis thaliana mutants for EXO84b are severely dwarfed and have compromised leaf epidermal cell and guard cell division. During cytokinesis, green fluorescent protein–tagged exocyst subunits SEC6, SEC8, SEC15b, EXO70A1, and EXO84b exhibit distinctive localization maxima at cell plate initiation and cell plate maturation, stages with a high demand for vesicle fusion. Finally, we present data indicating a defect in cell plate assembly in the exo70A1 mutant. We conclude that the exocyst complex is involved in secretory processes during cytokinesis in Arabidopsis cells, notably in cell plate initiation, cell plate maturation, and formation of new primary cell wall.     

Elektronenmikroskopische Untersuchungen von Differenzierungsvorgängen bei Moosen

Summary In meristematic cells of the gemma of Riella helicophylla and in young bud cells from the protonema of Funaria hygrometrica the cell plate is formed by fusion of small vesicles originating from the Golgi apparatus. These spherical vesicles of about 0.1 m diameter have an electron dense centre, probably consisting of pectic substances or their precursors. The endoplasmic reticulum producing multivesicular bodies participate in cell plate formation too. Another cytoplasmic component forming the cell plate are coated vesicles, the origin of which is the Golgi apparatus and perhaps also the endoplasmic reticulum. In view of these observations the question of whether the endoplasmic reticulum or the Golgi apparatus forms the cell plate must be answered in this way: both endoplasmic reticulum and Golgi apparatus supply material for growth of the cell plate. Multivesicular bodies, coated vesicles and other small vesicles of unknown nature participate in the formation of the primary wall.

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Zum Teil finanziert mit Sondermitteln des Landes Niedersachsen an Prof. Dr. M. Bopp.     

Cell Division in Gonyaulax catenella,A Marine Catenate Dinoflagellate*

Mitosis and cytokinesis are described and illustrated for the first time in the mesokaryotic, catenate dinoflagellate Gonyaulax catenella. A structure similar to the central body of G. tamarensis and G. monilata is shown by light and electron microscopy to be situated intranuclearly near the arms of the U-shaped interphase nucleus, and is suggested to function in the segregation of daughter chromosomes. This structure has the fine structure of a nucleolus, and it is suggested that the term central body be replaced by persistent nucleolus (= endosome). The time required for the completion of mitosis is 4–6 hr, while cytokinesis requires at least 2 hr. Cytokinesis begins during the mitotic cycle, and the plane of fission is perpendicular to the mitotic plane of division. Parental fission moieties are retained and shared by the daughter cells while either a new antero-sinistral moiety or a posterodextral moiety is synthesized by the dividing cell.     

Inhibition of very long acyl chain sphingolipid synthesis modifies membrane dynamics during plant cytokinesis

Plant cytokinesis requires intense membrane trafficking and remodeling to form a specific membrane structure, the cell plate that will ultimately separate the daughter cells. The nature and the role of lipids involved in the formation of the cell plate remain unclear. Plant membranes are particularly rich in sphingolipids such as glucosyl-ceramides with long (16 carbons) or very long (24 carbons) acyl chains. We reveal here that inhibition of the synthesis of sphingolipids with very long acyl chains induces defective cell plates with persistent vesicular structures and large gaps. Golgi-derived vesicles carrying material toward the cell plate display longer vesicle–vesicle contact time and their cargos accumulate at the cell plate, suggesting membrane fusion and/or recycling defects. In vitro fusion experiments between artificial vesicles show that glycosphingolipids with very long acyl chains stimulate lipid bilayer fusion. Therefore we propose that the very long acyl chains of sphingolipids are essential structural determinants for vesicle dynamics and membrane fusion during cytokinesis.     

The presence of extended phragmosomes containing cytoskeletal elements in fusiform cambial cells ofFraxinus excelsior L.

Summary Fusiform cambial cells of the ash (Fraxinus excelsior L.), which are strongly elongated and vacuolated, contain a phragmosome which traverses the whole length of the cells during preprophase and karyokinesis and which remains present during cytokinesis until it is integrated in cell plate with adjacent cytoplasm.The phragmosome consists of a thin perforated cytoplasmic layer located in the plane of the future cell plate. Otherwise oriented transvacuolar cytoplasmic layers or strands are not present in these cells.The phragmosome contains cytoskeletal elements, namely microtubules and also microfilament bundles both of which are oriented mainly in longitudinal direction.The phragmosomal microtubules are a new category of microtubules associated with cell division; presumably they guide the centrifugally growing cell plate to the parental cell wall site previously marked by the preprophase band of microtubules.     

Changes in the activity of the Golgi apparatus during the cell cycle in antheridial filaments ofChara vulgaris L.

Summary The number of dictyosomes found in one central cell section in antheridial filaments ofChara vulgaris increases proportionally to the cell length during interphase. The activity of Golgi apparatus was expressed by a number of Golgi vesicles surrounding a single dictyosome. These vesicles are most numerous during mitosis and cytokinesis,i.e., prior to and during cell plate formation. In the middle and late S phase the number of Golgi vesicles decreases by about 25%; subsequently, during the early and middle G₂, it increases again. At the end of the G₂ phase, Golgi vesicles are the scarcest.The increase in the number of Golgi vesicles during the G₂ phase coincides with the period of intense cellular elongation, and, thus, it is probably related to the enhanced synthesis of cell wall components.Coated vesicles are most numerous in prophase, metaphase, and early telophase, and during interphase in both late S and G₂ phase. It was found that the number of coated vesicles is proportional to the degree of condensation of nuclear chromatin.This work was supported by the Polish Academy of Sciences within the project 09.7.3.1.4.     

The KEULE gene is involved in cytokinesis in Arabidopsis

 We present evidence to show that the KEULE gene of Arabidopsis is involved in cytokinesis. Mutant keule embryos have large multinucleate cells with gapped or incomplete cross walls, as well as cell wall stubs that are very similar to those observed upon caffeine inhibition of cytokinesis in plants. These defects are observed in all populations of dividing cells in the mutant, including calli, but less frequently in mature cells. Cell division appears to be slowed down, and the planes of cell division are often misoriented. In late embryos and seedlings, cross-wall formation usually appears complete, suggesting that the requirement for KEULE during cytokinesis is not absolute. Nonetheless, keule mutants die as seedlings with large polyploid cells. The bloated surface layer of keule seedlings does not uniformly behave like wild-type epidermis, and patches of this layer assume characteristics of the underlying ground tissue. The cytokinesis defect of keule mutants may influence aspects of cellular differentiation. Received: 24 April 1996 / Accepted: 11 June 1996     

Cell division in caffeine-induced binucleate protonemal cells ofAdiantum. II. Formation of the preprophase band and cell division in centrifuged and non-centrifuged cells

Organization of microtubules (MTs) in relation to the behavior of nuclei was examined in dividing binucleate cells ofAdiantum capillus-veneris L. To induce binucleate cells, caffeine, an inhibitor of formation of the cell plate, was applied at 4 mM to synchronously dividing protonemal cells during cytokinesis (Murata and Wada 1993). Formation of the preprophase band (PPB) during the next cell cycle was examined in non-centrifuged and centrifuged cells. The two nuclei were separated or associated with one another in both non-centrifuged and centrifuged cells, although the location of the nuclei in the cylindrical protonemal cells was different (Murata and Wada 1993). Irrespective of centrifugation, a single PPB was formed around the nuclei in cells with associated nuclei. Two PPBs were formed in cells with separated nuclei in centrifuged cells. Patterns of mitosis and cytokinesis varied, depending on the location of the PPB and the distribution of the nuclei. The role of the nucleus in formation of the PPB is discussed.     

A biophysical model for plant cell plate maturation based on the contribution of a spreading force

Plant cytokinesis, a fundamental process of plant life, involves de novo formation of a “cell plate” partitioning the cytoplasm of dividing cells. Cell plate formation is directed by orchestrated delivery, fusion of cytokinetic vesicles, and membrane maturation to form a nascent cell wall by timely deposition of polysaccharides. During cell plate maturation, the fragile membrane network transitions to a fenestrated sheet and finally a young cell wall. Here, we approximated cell plate sub-structures with testable shapes and adopted the Helfrich-free energy model for membranes, including a stabilizing and spreading force, to understand the transition from a vesicular network to a fenestrated sheet and mature cell plate. Regular cell plate development in the model was possible, with suitable bending modulus, for a two-dimensional late stage spreading force of 2–6 pN/nm, an osmotic pressure difference of 2–10 kPa, and spontaneous curvature between 0 and 0.04 nm⁻¹. With these conditions, stable membrane conformation sizes and morphologies emerged in concordance with stages of cell plate development. To reach a mature cell plate, our model required the late-stage onset of a spreading/stabilizing force coupled with a concurrent loss of spontaneous curvature. Absence of a spreading/stabilizing force predicts failure of maturation. The proposed model provides a framework to interrogate different players in late cytokinesis and potentially other membrane networks that undergo such transitions. Callose, is a polysaccharide that accumulates transiently during cell plate maturation. Callose-related observations were consistent with the proposed model’s concept, suggesting that it is one of the factors involved in establishing the spreading force.

The late-stage onset of an “areal” spreading and stabilizing force is essential for regular plant cell plate development and maturation.     

Comparative effects of caffeine, its analogues and calcium deficiency on cytokinesis

The effects of caffeine, aminophylline, caffeic acid, and calcium deficiency on cytokinesis were studied by light and electron microscopy. All these treatments blocked cell plate formation, resulting in the formation of binucleate cells. The aggregation and organization of membranous vesicles at the ‘presumptive cell plate’ during these treatments appears similar to that of normal cells, but fusion of the vesicles is insufficient to form a complete cell plate. It is suggested that some aspect of membrane recognition and fusion is the process actually interfered with by these treatments. Greater numbers of binucleate cells and fewer partial cell plates were observed in cells treated with caffeine and aminophylline as compared with those exposed to caffeic acid or calcium deficiency, indicating that the latter treatments do not block cell plate formation as efficiently as the former.     

Dynamics of phragmoplastin in living cells during cell plate formation and uncoupling of cell elongation from the plane of cell division.

The cell plate is formed by the fusion of Golgi apparatus-derived vesicles in the center of the phragmoplast during cytokinesis in plant cells. A dynamin-like protein, phragmoplastin, has been isolated and shown to be associated with cell plate formation in soybean by using immunocytochemistry. In this article, we demonstrate that similar to dynamin, phragmoplastin polymerizes to form oligomers. We fused soybean phragmoplastin with the green fluorescence protein (GFP) and introduced it into tobacco BY-2 cells to monitor the dynamics of early events in cell plate formation. We demonstrate that the chimeric protein is functional and targeted to the cell plate during cytokinesis in transgenic cells. GFP-phragmoplastin was found to appear first in the center of the forming cell plate, and as the cell plate grew outward, it redistributed to the growing margins of the cell plate. The redistribution of phragmoplastin may require microtubule reorganization because the microtubule-stabilizing drug taxol inhibited phragmoplastin redistribution. Our data show that throughout the entire process of cytokinesis, phragmoplastin is concentrated in the area in which membrane fusion is active, suggesting that phragmoplastin participates in an early membrane fusion event during cell plate formation. Based on the dynamics of GFP-phragmoplastin, it appears that the process of cell plate formation is completed in two phases. The first phase is confined to the cylinder of the phragmoplast proper and is followed by a second phase that deposits phragmoplast vesicles in a concentric fashion, resulting in a ring of fluorescence, with the concentration of vesicles being higher at the periphery. In addition, overexpression of GFP-phragmoplastin appears to act as a dominant negative, slowing down the completion of cell plate formation, and often results in an oblique cell plate. The latter appears to uncouple cell elongation from the plane of cell division, forming twisted and elongated cells with longitudinal cell divisions.     

Profilin 1 is required for abscission during late cytokinesis of chondrocytes

Profilins are key factors for dynamic rearrangements of the actin cytoskeleton. However, the functions of profilins in differentiated mammalian cells are uncertain because profilin deficiency is early embryonic lethal for higher eukaryotes. To examine profilin function in chondrocytes, we disrupted the profilin 1 gene in cartilage (Col2pfn1). Homozygous Col2pfn1 mice develop progressive chondrodysplasia caused by disorganization of the growth plate and defective chondrocyte cytokinesis, indicated by the appearance of binucleated cells. Surprisingly, Col2pfn1 chondrocytes assemble and contract actomyosin rings normally during cell division; however, they display defects during late cytokinesis as they frequently fail to complete abscission due to their inability to develop strong traction forces. This reduced force generation results from an impaired formation of lamellipodia, focal adhesions and stress fibres, which in part could be linked to an impaired mDia1‐mediated actin filament elongation. Neither an actin nor a poly‐proline binding‐deficient profilin 1 is able to rescue the defects. Taken together, our results demonstrate that profilin 1 is not required for actomyosin ring formation in dividing chondrocytes but necessary to generate sufficient force for abscission during late cytokinesis.