Visualization of actin cytoskeletal dynamics during the cell cycle in tobacco (Nicotiana tabacum L. cv Bright Yellow) cells

BACKGROUND INFORMATION: The actin cytoskeleton forms distinct actin arrays which fulfil their functions during cell cycle progression. Reorganization of the actin cytoskeleton occurs during transition from one actin array to another. Although actin arrays have been well described during cell cycle progression, the dynamic organization of the actin cytoskeleton during actin array transition remains to be dissected. RESULTS: In the present study, a GFP (green fluorescent protein)-mTalin (mouse talin) fusion gene was introduced into suspension-cultured tobacco BY-2 (Nicotiana tabacum L. cv Bright Yellow) cells by a calli-cocultivation transformation method to visualize the reorganization of the actin cytoskeleton in vivo during the progression of the cell cycle. Typical actin structures were indicated by GFP-mTalin, such as the pre-prophase actin band, mitotic spindle actin filament cage and phragmoplast actin arrays. In addition, dynamic organization of actin filaments was observed during the progression of the cell from metaphase to anaphase. In late metaphase, spindle actin filaments gradually shrank to the equatorial plane along both the long and short axes. Soon after the separation of sister chromosomes, actin filaments aligned in parallel at the cell division plane, forming a cylinder-like structure. During the formation of the cell plate, one cylinder-like structure changed into two cylinder-like structures: the typical actin arrays of the phragmoplast. However, the two actin arrays remained overlapping at the margin of the centrally growing cell plate, forming an actin wreath. When the cell plate matured further, an actin filament network attached to the cell plate was formed. CONCLUSIONS: Our results clearly describe the dynamic organization of the actin cytoskeleton during mitosis and cytokinesis of a plant cell. This demonstrates that GFP-mTalin-transformed tobacco BY-2 cells are a valuable tool to study actin cytoskeleton functions in the plant cell cycle.     

The dynamics of the actin cytoskeleton during sporogenesis in Psilotum nudum L.

The actin cytoskeleton (microfilaments, MFs) accompanies the tubulin cytoskeleton (microtubules) during the meiotic division of the cell, but knowledge about the scope of their physiological competence and cooperation is insufficient. To cast more light on this issue, we analysed the F-actin distribution during the meiotic division of the Psilotum nudum sporocytes. Unfixed sporangia of P. nudum were stained with rhodamine-phalloidin and 4′,6-diamidino-2-phenylindole dihydrochloride, and we monitored the changes in the actin cytoskeleton and nuclear chromatin throughout sporogenesis. We observed that the actin cytoskeleton in meiotically dividing cells is not only part of the kariokinetic spindle and phragmoplast but it also forms a well-developed network in the cytoplasm present in all phases of meiosis. Moreover, in telophase I F-actin filaments formed short-lived phragmoplast, which was adjacent to the plasma membrane, exactly at the site of future cell wall formation. Additionally, the meiocytes were pre-treated with cytochalasin-B at a concentration that causes damage to the MFs. This facilitated observation of the effect of selective MFs damage on the course of meiosis and sporogenesis of P. nudum. Changes were observed that occurred in the cytochalasin-treated cells: the daughter nuclei were located abnormally close to each other, there was no formation of the equatorial plate of organelles and, consequently, meiosis did not occur normally. It seems possible that, if the actin cytoskeleton only is damaged, regular cytokinesis will not occur and, hence, no viable spores will be produced.     

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

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

Chaotization of division spindle, phragmoplast, and telophase chromosome groups in wheat x wheatgrass F1 hybrids meiosis

The paper describes the phenomenon of disorganization of completely formed subcellular structures: division spindle, phragmoplast and chromosome telophase groups. These structures disintegrate into their elements (cytoskeletal fibers, chromosomes) that transform into chaotic system. Chaotization of cytoskeleton structures such as prophase spindle in mitosis or perinuclear ring in meiosis is a normal step of wild type plant cell division. Disintegration of division spindle and phragmoplast presumably indicate the abnormality of temporal regulation of cytoskeleton cycle during meiosis. Disintegration of telophase chromosome groups and the migration of the chromosomes backward to the equatorial area might mean the abnormal start of some prometaphase mechanisms, in particular, chromokinesins activation.     

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.     

Disruption of male meiosis in transgenic line tobacco res91

The course of meiosis in male-sterile trasgenic tobacco line res91 has been analysed. Cytological analysis included visualization of the spindle and phragmoplast. Abnormal meiosis was characterized with 1) cytomixis; 2) deformation of nuclei at prophase 2 in part of the cells, and lack of spindle formation in such cells at M2; 3) desorientation of spindles in meiosis 2. This set of abnormalities allows to suppose certain disturbancies of cytoskeleton during male meiosis in res91.     

Plant actin filament and microtubule interactions during anaphase--telophase transition: effects of antagonist drugs

F-actin and microtubule co-distribution and interaction were studied during anaphase-telophase. Rapid and drastic changes in the cytoskeleton during these particular stages were studied in isolated plant endosperm cells of the blood lily. These wall-free cells can be considered as natural dividing protoplasts. As identified previously, an F-actin cytoskeletal network characterized the plant cortex and formed an elastic cage around the spindle, remaining throughout interphase, mitosis and cytokinesis. Actin was specifically labeled by fluorescent phalloidin and/or monoclonal antibodies. Gold-labelled secondary antibodies were used for ultrastructural observations and silver-enhancement was applied for video-enhanced microscopy. Microtubule and microfilament dynamics and interaction were studied using drug antagonists to actin (cytochalasins B, D) and to tubulin (colchicine). This permitted precise correlations to be made between chromosome movement inhibition and alteration in the actin/tubulin cytoskeleton. During anaphase chromosome migration, the cortical actin network was stretched along the microtubular spindle, while it remained homogeneous when anaphase was inhibited by colchicine. Cytochalasins did not inhibit chromosome movement but altered actin distribution. A new population of actin filaments appeared at the equator in late anaphase before the microtubular phragmoplast was formed and contributed to cell plate formation. Our conclusion is that F-actin-microtubule interaction may contribute to the regulatory mechanism of plant cytokinesis.     

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.     

Displacement of the mitotic apparatuses by centrifugation reveals cortical actin organization during cytokinesis in cultured tobacco BY-2 cells

In plant cytokinesis, actin is thought to be crucial in cell plate guidance to the cortical division zone (CDZ), but its organization and function are not fully understood. To elucidate actin organization during cytokinesis, we employed an experimental system, in which the mitotic apparatus is displaced and separated from the CDZ by centrifugation and observed using a global–local live imaging microscope that enabled us to record behavior of actin filaments in the CDZ and the whole cell division process in parallel. In this system, returning movement of the cytokinetic apparatus in cultured-tobacco BY-2 cells occurs, and there is an advantage to observe actin organization clearly during the cytokinetic phase because more space was available between the CDZ and the distantly formed phragmoplast. Actin cables were clearly observed between the CDZ and the phragmoplast in BY-2 cells expressing GFP-fimbrin after centrifugation. Both the CDZ and the edge of the expanding phragmoplast had actin bulges. Using live-cell imaging including the global–local live imaging microscopy, we found actin filaments started to accumulate at the actin-depleted zone when cell plate expansion started even in the cell whose cell plate failed to reach the CDZ. These results suggest that specific accumulation of actin filaments at the CDZ and the appearance of actin cables between the CDZ and the phragmoplast during cell plate formation play important roles in the guidance of cell plate edges to the CDZ.     

Vesicular transport protein Arf6 modulates cytoskeleton dynamics for polar body extrusion in mouse oocyte meiosis

Arf6 (ADP-ribosylation factor 6) is known to play important roles in membrane dynamics through the regulation of actin filament reorganization for multiple cellular processes such as cytokinesis, phagocytosis, cell migration and tumor cell invasion. However, the functions of Arf6 in mammalian oocyte meiosis have not been clarified. In present study we showed that Arf6 expressed in mouse oocytes and was mainly distributed around the spindle during meiosis. Depletion of Arf6 by morpholino microinjection caused oocytes failing to extrude first polar body. Further analysis indicated that Arf6 knock down caused the aberrant actin distribution, which further induced the failure of meiotic spindle movement. And the loss of oocyte polarity also confirmed this. The regulation of Arf6 on actin filaments in mouse oocytes might be due to its effects on the phosphorylation level of cofilin and the expression of Arp2/3 complex. Moreover, we found that the decrease of Arf6 caused the disruption of spindle formation, indicating the multiple roles of Arf6 on cytoskeleton dynamics in meiosis. In summary, our results indicated that Arf6 was involved in mouse oocyte meiosis through its functional roles in actin-mediated spindle movement and spindle organization.     

Dynamics of microtubular cytoskeleton during meiosis in higher plants. VIII. Comparison between successive and simultaneous cytokinesis

The abnormal cytoskeleton cycle in meiosis in pollen mother cells of cereal wide hybrids F reveals the role of polar microtubules in phragmoplast formation during successive cytokinesis. The cytoskeletal rearrangements during successive and simultaneous cytokinesis in higher plant meiosis are compared.     

Microtubule organization during successive microsporogenesis in Allium cepa and simultaneous cytokinesis in Nicotiana tabacum

Microtubule cytoskeleton organization during microspore mother cell (MMC) meiosis in Allium cepa L. and microsporogenesis in Nicotiana tabacum L. was examined. The MMC microtubules (MTs) were short and well dispersed in the cytoplasm of both taxa. As the MMCs of both species entered metaphase of meiosis I, the MTs constructed a spindle that facilitated the chromosomes to orient in the meridian plane. At anaphase of meiosis I, the spindle MTs differentiated into two types: one MT type became short, pulled the chromosomes toward the two poles, and was designated as centromere MTs; the second type of MT connected the two poles, and was designated as pole MTs. In A. cepa, where successive cytokinesis was observed, pole MTs assumed a tubbish shape. Some new short MTs aggregated in the meridian plane and constricted to form a phragmoplast, which developed into a cell plate, divided the cytoplasm into two parts and produced a dyad. However, in tobacco, a phragmoplast was not generated in anaphase of meiosis I and II and cytokinesis did not occur. The spindle MTs depolymerized and reorganized the radial arrangement of MTs from the nucleate surface to the periplasm during anaphase. Following telophase of meiosis II, the cytoplasm produced centripetal furrows, which met in the center of the cell and divided it into four parts, serving as a form of cytokinesis. In this process, MTs appeared to bear no relationship to cytokinesis.     

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

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

Conformation of cytoskeletal elements during the division of infected Lupinus albus L. nodule cells

Lupin nodule cells maintain their ability to divide for several cycles after being infected by endosymbiotic rhizobia. The conformation of the cytoskeletal elements of nodule cells was studied by fluorescence labelling, immunocytochemistry, and laser confocal and transmission electron microscopy. The dividing infected cells showed the normal microtubule and actin patterns of dividing plant cells. The clustered symbiosomes were tethered to the spindle-pole regions and moved to the cell poles during spindle elongation. In metaphase, anaphase, and early telophase, the symbiosomes were found at opposite cell poles where they did not interfere with the spindle filaments or phragmoplast. This symbiosome positioning was comparable with that of the organelles (which ensures organelle inheritance during plant cell mitosis). Tubulin microtubules and actin microfilaments appeared to be in contact with the symbiosomes. The possible presence of actin molecular motor myosin in nodules was analysed using a monoclonal antibody against the myosin light chain. The antigen was detected in protein extracts of nodule and root cytosol as bands of approximately 20 kDa (the size expected). In the nodules, an additional polypeptide of 65 kDa was found. Immunogold techniques revealed the antigen to be localized over thin microfilaments linked to the cell wall, as well as over the thicker microfilament bundles and surrounding the symbiosomes. The pattern of cytoskeleton rearrangement in dividing infected cells, along with the presence of myosin antigen, suggests that the positioning of symbiosomes in lupin nodule cells might depend on the same mechanisms used to partition genuine plant cell organelles during mitosis.     

The actin cytoskeletal characteristics of the root meristem cells in Beta vulgaris L. at different mitotic stages

For the first time the actin cytoskeleton in meristem cells of B. vulgaris root has been studied. It was established that all discovered earlier patterns of microfilament arrangement were also specific for meristem of present species. Specific rearrangements of actin in the sites of spindle (prophase) and phragmoplast formation (telophase) were revealed. It is supposed, that actin cytoskeleton determines certain position of spindle in cells and takes part in the cell plate formation.     

Formation and function of phragmoplast during successive cytokinesis stages in higher plant meiosis

Cytoskeletal rearrangements were studied during meiotic telophase in a number of monocotyledonous plant species. Wild type and abnormal meiosis (in wide cereal hybrids, meiotic mutants and allolines) was analyzed. It was found that central spindle fibers that move centrifugally, along with newly-formed MTs, are the basis of phragmoplast formation and function in PMCs of monocotyledonous plant species with successive cytokinesis stages. A model for centrifugal movement of the meiotic phragmoplast is proposed; this model is a modification of the corresponding process during B-anaphase.     

Abundance of actin filaments in the preprophase band and mitotic spindle of brick1 Zea mays mutant

The preprophase band and mitotic spindle of dividing protodermal cells of wild-type Zea mays leaves include few actin filaments. Surprisingly, abundant actin filaments were observed in the above arrays, in dividing protodermal cells in the leaves of the brick1 mutant. The same abundance was observed in the spindle of Taxol-treated brick1 mitotic protodermal cells. Apart from the above difference, the relevant arrays displayed normal microtubule organization in both wild type and mutant cells, as far as can be discerned by immunofluorescence microscopy. Accordingly, the abundance of actin filaments in the preprophase band and spindle of brick1 mitotic cells seems not to influence the structure of the above arrays and might be a non-functional “side-effect” of defective F-actin organization in this mutant.     

Localization of actin filaments on mitotic apparatus in tobacco BY-2 cells

Actin filaments are among the major components of the cytoskeleton, and participate in various cellular dynamic processes. However, conflicting results had been obtained on the localization of actin filaments on the mitotic apparatus and their participation in the process of chromosome segregation. We demonstrated by using rhodamine-phalloidin staining, the localization of actin filaments on the mitotic spindles of tobacco BY-2 cells when the cells were treated with cytochalasin D. At prophase, several clear spots were observed at or near the kinetochores of the chromosomes. At anaphase, the actin filaments that appeared to be pulling chromosomes toward the division poles were demonstrated. However, as there was a slight possibility that these results might have been the artifacts of cytochalasin D treatment or the phalloidin staining, we analyzed the localization of actin filaments at the mitotic apparatus immunologically. We cloned a novel BY-2 -type actin cDNA and prepared a BY-2 actin antibody. The fluorescence of the anti-BY-2 actin antibody was clearly observed at the mitotic apparatus in both non-treated and cytochalasin D-treated BY-2 cells during mitosis. The facts that similar results were obtained in both actin staining with rhodamine-phalloidin and immunostaining with actin antibody strongly indicate the participation of actin in the organization of the spindle body or in the process of chromosome segregation. Furthermore, both filamentous actin and spindle bodies disappeared in the cells treated with propyzamide, which depolymerizes microtubules, supporting the notion that actin filaments are associated with microtubules organizing the spindle body.Hiroshi Yasuda and Katsuhiro Kanda contributed equally.     

黄蝉和姜花生殖细胞内肌动蛋白微丝的定位

用荧光标记的鬼笔碱染色,对离体的黄蝉和姜花的生殖细胞内肌动蛋白微丝的分布进行了研究,结果证明两种植物的生殖细胞内部都存在一个微丝网络,黄蝉生殖细胞的比姜花的简单,微丝束较粗。但姜花生殖细胞的网络微丝束比黄蝉的更紧密地环绕着核。用免疫荧光技术在黄蝉生殖细胞的分裂前期和中期,可以观察到一些微丝束的存在,但在分裂后期和末期细胞内的肌动蛋白则变为颗粒状。