Hiroh Shibaoka

Actin filaments and microtubules in the preprophase band and phragmoplast of tobacco cells.

SummaryTreatment with lysine prior to fixation of tobacco BY-2 cells with formaldehyde improved the preservation of actin filaments in the cells and enabled us to observe both networks of actin filaments and microtubules in the same cells. By using this method, we observed that (1) actin filaments were present in the preprophase band; (2) the actin filaments in the preprophase band and phragmoplast were runnig in the same direction as the microtubules in their respective structures; (3) a cortical network of actin filaments was present throughout all stages of cell cycle.The present method did not preserve the cortical actin filaments in interphase cells. The procedure for staining microtubules destroyed them.

Cytoskeletal Ultrastructure of Phragmoplast-Nuclei Complexes Isolated from Cultured Tobacco Cells

A preparation of phragmoplasts with a purity of about 40% was isolated from tobacco BY-2 cells whose cell cycle was synchronized by treatment with aphidicolin and propyzamide. Each isolated phragmoplast was associated with the daughter nuclei and the association was maintained after the phragmoplast lost either its actin filaments or its microtubules. Actin filaments identified by heavy meromyosin arrowhead decoration were abundant in the isolated phragmoplasts. Most of the filaments were oriented perpendicularly or nearly perpendicularly to the equatorial plate and in about 80% of the filaments thus oriented heavy meromyosin arrowheads pointed away from the plate. Abundant vesicles were present in the isolated phragmoplast-nuclei complex and they were associated with phragmoplast microtubules.

Interrelation between the spatial disposition of actin filaments and microtubules during the differentiation of tracheary elements in culturedZinnia cells

SummaryChanges in the spatial relationship between actin filaments and microtubules during the differentiation of tracheary elements (TEs) was investigated by a double staining technique in isolatedZinnia mesophyll cells. Before thickening of the secondary wall began to occur, the actin filaments and microtubules were oriented parallel to the long axis of the cell. Reticulate bundles of microtubules and aggregates of actin filaments emerged beneath the plasma membrane almost simultaneously, immediately before the start of the deposition of the secondary wall. The aggregates of actin filaments were observed exclusively between the microtubule bundles. Subsequently, the aggregates of actin filaments extended preferentially in the direction transverse to the long axis of the cell, and the arrays of bundles of microtubules which were still present between the aggregates of actin filaments became transversely aligned. The deposition of the secondary walls then took place along the transversely aligned bundles of microtubules.Disruption of actin filaments by cytochalasin B produced TEs with longitudinal bands of secondary wall, along which bundles of microtubules were seen, while TEs produced in the absence of cytochalasin B had transverse bands of secondary wall. These results indicate that actin filaments play an important role in the change in the orientation of arrays of microtubules from longitudinal to transverse. Disruption of microtubules by colchicine resulted in dispersal of the regularly arranged aggregates of actin filaments, but did not inhibit the formation of the aggregates itself, suggesting that microtubules are involved in maintaining the arrangement of actin filaments but are not involved in inducing the formation of the regularly arranged aggregates of actin filaments.These findings demonstrate that actin filaments cooperate with microtubules in controlling the site of deposition of the secondary wall in developing TEs.

Stabilization of cortical microtubules by the cell wall in cultured tobacco cells : Effects of extensin on the cold-stability of cortical microtubules.

Cortical microtubules (MTs) in protoplasts prepared from tobacco (Nicotiana tabacum L.) BY-2 cells were found to be sensitive to cold. However, as the protoplasts regenerated cell walls they became resistant to cold, indicating that the cell wall stabilizes cortical MTs against the effects of cold. Since poly-l-lysine was found to stabilize MTs in protoplasts, we examined extensin, an important polycationic component of the cell wall, and found it also to be effective in stabilizing the MTs of protoplasts. Both extensin isolated from culture filtrates of tobacco BY-2 cells and extensin isolated in a similar way from cultures of tobacco XD-6S cells rendered the cortical MTs in protoplasts resistant to cold. Extensin at 0.1 mg·ml−1 was as effective as the cell wall in this respect. It is probable that extensin in the cell wall plays an important role in stabilizing cortical MTs in tobacco BY-2 cells.

Gibberellin stabilizes microtubules in onion leaf sheath cells

SummaryColchicine and cremart (O-ethyl O-(3-methyl-6-nitrophenyl) N-sec-butylphosphorothioamidate) disrupt microtubules in leaf sheath cells of onion plants (Allium cepa L. cv. Senshu-Chuko) and cause cell swelling to make the basal parts of the plants bulbous. Gibberellin A3(GA3) protects microtubules from disruption by colchicine and cremart and suppresses the swelling caused by them. GA3 also protects microtubules from disruption by low temperature.

The cyclic reorientation of cortical microtubules in epidermal cells of azuki bean epicotyls: the role of actin filaments in the progression of the cycle

Abstract. The orientation of microtubules (MTs) was examined in epidermal cells of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls. The orientation of MTs adjacent to the outer tangential wall of the cells, which has a crossed polylamellate structure with lamellae of longitudinal cellulose microfibrils alternating with lamellae of transverse cellulose microfibrils, differed from one cell to another. Treatment with an auxin-free solution caused the accumulation of cells with longitudinal MTs and subsequent treatment with a solution that contained auxin resulted in the accumulation of cells with transverse MTs, showing that sequential treatments with auxin-free and auxin-containing solutions can synchronize the reorientation of MTs. The MTs, once reoriented from longitudinal to transverse, returned to longitudinal and then back to transverse once again, the duration of the cycle being about 6 h. Gibberellic acid, known to increase the percentage of cells with transverse MTs, promoted reorientation of MTs from longitudinal to transverse and inhibited that from transverse to longitudinal. Cytochalasin D, an agent that disrupts actin filaments, speeded up the reorientation from transverse to longitudinal and slowed down that from longitudinal to transverse. It caused an increase in the percentage of cells with MTs in mixed orientation, and the percentage of such cells was highest when the percentage of cells with longitudinal MTs was decreasing and that of cells with transverse MTs was increasing.

Effects of abscisic acid on the orientation and cold stability of cortical microtubules in epicotyl cells of the dwarf pea

SummaryThe effects of abscisic acid (ABA) on the orientation and cold stability of cortical microtubules (MTs) in epidermal cells of epicotyls of the dwarf pea,Pisum sativum L. cv. Little Marvel, were examined by immunofluorescence microscopy. The effect of ABA on the elongation of epicotyls and on the orientation of cortical MTs was opposite to that of gibberellin A3 (GA3). Treatment with ABA, which reduced the promotion of epicotyl elongation by GA3, eliminated the GA3-induced predominance of transverse MTs and resulted in a predominance of longitudinal MTs. The effect of ABA on the cold stability of cortical MTs was also opposite to that of GA3. ABA increased the cold stability of MTs, while GA3 decreased it. The predominance of longitudinal MTs brought about by ABA may have some relationship to ABA-induced inhibition of the elongation of the epicotyl. ABA may alter membrane proteins to stabilize cortical MTs and induce cold hardiness of plants.

The plant cytoskeleton.

Particles that can nucleate microtubules in vitro have been isolated from higher plant cells. Observations of living cells injected with fluorescent probes have improved our understanding of plant cytoskeleton dynamics. Despite growing recognition of the need for biochemical studies on cytoskeleton-associated proteins, little progress has been made in this field in the past year, although plant lamins have been isolated and partially characterized.

Reorganization of actin filaments associated with the differentiation of tracheary elements inZinnia mesophyll cells

The differentiation of tracheary elements is a dramatic example ofcytodifferentiation in higher plants (ToRREu etal. 1971, ROBERTS 1976, FUKUDA and KOMAMINv 1985). It has been suggested that, during this differentiation, the rearrangement of microtubules occurs preceding the localized thickening of secondary wall and the orientation of the microtubules determines the pattern of bands of the secondary walls (FALCONER and SEA6ULL 1985, 1986). However, there have been no reports of changes in the arrangement of actin filaments during the differentiation of tracheary elements, even though these filaments are essential components of the cytoskeleton. However, QUADER etal. (1986) reported the presence of actin-like filaments in Cobaea seed hair cells in which localized thickening of cell walls occurs. We have been able to demonstrate, for the first time, a dynamic change in the arrangement of actin filaments during the differentiation of tracheary elements, and we have also found an orderly array of loci for the organization of actin filaments. Our novel findings are described in this paper. Single cells were isolated from the mesophyll of the first leaves of 14-day-old seedlings of Zinnia elegans, and were cultured for 75 hours in the presence of 0.1 mg/ml

The cyclic reorientation of cortical microtubules on walls with a crossed polylamellate structure: effects of plant hormones and an inhibitor of protein kinases on the progression of the cycle

SummaryThe outer tangential wall (OTW) of epidermal cells of azuki bean epicotyls has a crossed polylamellate structure, in which lamellae of longitudinal cellulose microfibrils alternate with lamellae of transverse cellulose microfibrils. This implies that the cyclic reorientation of cortical microtubules (MTs) from longitudinal to transverse and from transverse to longitudinal occurs on the OTW. Treatment with a solution that contained no auxin caused the accumulation of cells with longitudinal MTs, suggesting that auxin is required for the reorientation of MTs from longitudinal to transverse during the reorientation cycle. Treatment with 6-dimethylaminopurine (DMAP), an inhibitor of protein kinases that promoted the reorientation of MTs from transverse to longitudinal, resulted in the accumulation of cells with longitudinal MTs. Subsequent treatment with auxin caused a marked increase in the percentage of cells with transverse MTs and then a decrease in the percentage, indicating that the reorientation of MTs from longitudinal to transverse and then from transverse to longitudinal occurred during treatment with auxin. The percentage of cells with transverse MTs decreased more slowly in segments that had been pretreated with gibberellin A3 (GA) than in segments that had been pretreated without GA, suggesting that GA, in cooperation with auxin, caused the suppression of the reorientation of MTs from transverse to longitudinal.