Inhibition of cell-plate formation by brefeldin A inhibited the depolymerization of microtubules in the central region of the phragmoplast.

Treatment of tobacco BY-2 cells with 20 microM brefeldin A (BFA), which causes disassembly of the Golgi apparatus (Yasuhara et al. 1995), completely inhibited the formation of the cell plate when the treatment was started before the chromosomes had begun to to condense. In cells in which cell-plate formation was inhibited by BFA, the centrifugal development of the phragmoplast was also inhibited. In such cells, the depolymerization of microtubules in the central region of the phragmoplast did not occur at least for 1 h after the formation of the phragmoplast, while the centrifugal development of the phragmoplast and cell-plate formation were completed in almost all cells not treated with BFA. The inhibition of cell-plate formation seems to inhibit the centrifugal development of the phragmoplast by inhibiting the depolymerization of microtubules in the central region of the phragmoplast, which is required for the supply of free tubulin necessary for the polymerization of microtubules at the outer margins of the phragmoplast.

Plasma membrane-associated actin in bright yellow 2 tobacco cells. Evidence for interaction with microtubules

Plasma membrane ghosts form when plant protoplasts attached to a substrate are lysed to leave a small patch of plasma membrane. We have identified several factors, including the use of a mildly acidic actin stabilization buffer and the inclusion of glutaraldehyde in the fixative, that allow immunofluorescent visualization of extensive cortical actin arrays retained on membrane ghosts made from tobacco (Nicotiana tabacum L.) suspension-cultured cells (line Bright Yellow 2). Normal microtubule arrays were also retained using these conditions. Membrane-associated actin is random; it exhibits only limited coalignment with the microtubules, and microtubule depolymerization in whole cells before wall digestion and ghost formation has little effect on actin retention. Actin and microtubules also exhibit different sensitivities to the pH and K+ and Ca2+ concentrations of the lysis buffer. There is, however, strong evidence for interactions between actin and the microtubules at or near the plasma membrane, because both ghosts and protoplasts prepared from taxol-pretreated cells have microtubules arranged in parallel arrays and an increased amount of actin coaligned with the microtubules. These experiments suggest that the organization of the cortical actin arrays may be dependent on the localization and organization of the microtubules.

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

The orientation of microtubules (MTs) was examined in epidermal cells of azuki bean (Vigna angular is 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 6h. 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.

Gibberellin A3 causes a decrease in the accumulation of mRNA for ACC oxidase and in the activity of the enzyme in azuki bean (Vigna angularis) epicotyls.

Differential screening, aimed at the isolation of cDNA clones of mRNAs whose accumulation is influenced by GA3, resulted in the isolation of a cDNA clone of an mRNA whose level was decreased by GA3 in segments of epicotyls of Vigna angularis. The putative protein encoded by this cDNA resembled the 1-aminocyclopropane-1-carboxylate oxidases (ACC oxidases) identified in other plant species (about 80% homology at the amino acid level). Thus, the corresponding gene was designated AB-ACO1 (azuki bean ACC oxidase). GA3 also decreased the activity of ACC oxidase in azuki bean epicotyls, but it did not decrease the rate of ethylene evolution. In fact, GA3 increased the rate of ethylene evolution and the level of ACC. Thus, GA3 seemed to increase the production of ethylene by promoting the synthesis of ACC.

TKRP125, a kinesin-related protein involved in the centrosome-independent organization of the cytokinetic apparatus in tobacco BY-2 cells.

Analysis of a cDNA for a 125 kDa polypeptide, previously isolated from phragmoplasts of tobacco BY-2 cells as a candidate for a plus end-directed microtubule motor, revealed this polypeptide to be a novel member of the kinesin superfamily. We named this protein TKRP125 (tobacco kinesin-related polypeptide of 125 kDa). The strong similarity between TKRP125 and members of the bimC subfamily in terms of the amino acid sequence of the amino-terminal motor domain indicated that TKRP125 belonged to the bimC subfamily. An antibody against a short peptide from the motor domain of TKRP125 inhibited the GTP- or ATP-dependent translocation of phragmoplast microtubules in membrane-permeabilized BY-2 cells, suggesting a role for TKRP125 in microtubule translocation, which is considered to be involved in the formation and/or maintenance of the bipolar structure of the phragmoplast. The expression of TKRP125 was found to be cell cycle-dependent. TKRP125 was not present in cells at the G1 phase. It began to appear at the S phase and accumulated during the G2 phase. The distribution of TKRP125 changed as the arrangement of microtubules changed with the progression of the cell cycle. TKRP125 was distributed along cortical microtubules during the S phase and along microtubules in the preprophase band and perinuclear microtubules in premitotic cells. It was also present in the nucleus in premitotic cells. In cells in M phase, TKRP125 was distributed along spindle microtubules. It accumulated at the equatorial plane of the spindle as the spindle elongated. In cytokinetic cells, TKRP125 was colocalized with phragmoplast microtubules. These observations suggest the possible involvement of TKRP125 in the cell cycle-dependent changes in arrays of microtubules, including the organization of the phragmoplast, and in the movement of chromosomes in anaphase cells.

Effects of brefeldin A on the formation of the cell plate in tobacco BY-2 cells.

Treatment with 20 microM brefeldin A (BFA) for 60 min caused the disassembly of the Golgi apparatus in tobacco BY-2 cells, and the effect of BFA was reversible. Connections between Golgi cisternae and the ER were observed in cells that had been treated for 15 min with BFA. BFA applied to cells at metaphase allowed the cells to form aniline blue-positive cell plates but not to complete cytokinesis. BFA seems to inhibit cytokinesis by shutting off the supply of cell-plate materials by disassembling the Golgi apparatus.

Isolation of polypeptides with microtubule-translocating activity from phragmoplasts of tobacco BY-2 cells.

As part of our efforts to understand the molecular basis of the microtubule-associated motility that is involved in cytokinesis in higher plant cells, an attempt was made to identify proteins with the ability to translocate microtubules in an extract from isolated phragmoplasts. Homogenization of isolated phragmoplasts in a solution that contained MgATP, MgGTP and a high concentration of NaCl resulted in the release from phragmoplasts of factors with ATPase and GTPase activity that were stimulated by microtubules. A protein fraction with microtubule-dependent ATPase and GTPase activity caused minus-end-headed gliding of microtubules in the presence of ATP or GTP. Polypeptides with microtubule-translocating activity cosedimented with microtubules that had been assembled in vitro from brain tubulin and were dissociated from sedimented microtubules by addition of ATP or GTP. After cosedimentation and dissociation procedures, a 125 kDa polypeptide and a 120 kDa polypeptide were recovered in a fraction that supported minus-end-headed gliding of microtubules. The rate of microtubule gliding that was caused by the fraction that contained the 125 kDa and 120 kDa polypeptides as main components was 1.28 microns/minute in the presence of ATP and 0.50 microns/minute in the presence of GTP. This fraction contained some microtubule-associated polypeptides in addition to the 125 kDa and 120 kDa polypeptides, but a fraction that contained only these additional polypeptides did not cause any translocation of microtubules. Thus, it appeared that the 125 kDa and 120 kDa polypeptides were responsible for translocation of microtubules. These polypeptides with plus-end-directed motor activity may play an important role in formation of the cell plate and in the organization of the phragmoplast.

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.

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.

[The effect of new broad-spectrum cephamycin, cefotetan administered intravenously on human fecal flora (author's transl)].

The changes in the fecal flora and their correlation with the fecal drug concentrations were studied, employing 4 volunteers (healthy adult males) to whom 1 g of cefotetan was intravenously administered daily for 6 days. 1. In all of the subjects, the total counts of fecal organisms were slightly decreased 1 day after administration of cefotetan, then showed gradual increase, and recovered to the normal level on 8 days after the withdrawal of the drug. 2. Cefotetan caused drastic suppression of normal aerobic and anaerobic flora during the administration period, except streptococci that remained unchanged at all. The bacterial groups suppressed were bacteroidaceae, eubacteria, bifidobacteria, peptococcaceae, lactobacilli, enterobacteriaceae, lecithinase-positive clostridia, and staphylococci. Yeasts were found to increase during the administration period. 3. The changes in flora well correlated with the fecal level of the drug concentrations. This is in accordance with the in vitro antibacterial activity of the drug. 4. A period of more than 15 days was required after the withdrawal of cefotetan to recover the normal fecal flora. 5. Loss of body weight and diarrhoea were not observed in any of the subjects.

Isolation of plant tubulin from azuki bean epicotyls by ethyl N-phenylcarbamate-Sepharose affinity chromatography.

A new method for isolating plant tubulin was devised. The method involves affinity chromatography with ethyl N-phenylcarbamate and Sepharose. The protein isolated by the affinity chromatography and purified by two successive chromatographical steps on DEAE-Sephadex A-50 and Sephadex G-200 consisted of two subunits which possessed the same mobilities as the alpha- and beta-subunits of rabbit brain tubulin on SDS-polyacrylamide gel electrophoresis and showed colchicine-binding activity.

Changes in microfibril arrangement on the inner surface of the epidermal cell walls in the epicotyl of Vigna angularis ohwi et ohashi during cell growth.

Throughout the entire period of cell growth, the microfibrils on the inner surface of the outer tangential walls of the epidermal cells of Vigna angularis epicotyls are running parallel to one another and their orientation differs from cell to cell. Although transverse, oblique and longitudinal microfibrils can be observed irrespective of cell age, the frequency distribution of microfibril orientation changes with age. In young cells, transversely oriented microfibrils predominate. In cells of medium age, which are still undergoing elongation, transverse, oblique and longitudinal microfibrils are present in quite similar frequencies. In old, non-growing cells, longitudinally oriented microfibrils are predominent. A decrease in the relative frequency of transversely oriented microfibrils with cell age was also observed in the radial epidermal walls.

Effects of gibberellin and colchicine on microfibril arrangement in epidermal cell walls of Vigna angularis Ohwi et Ohashi epicotyls.

Gibberellic-acid (GA3) treatment of azukibean epicotyls resulted in alterations of the direction of newly deposited microfibrils, on the cell walls. Cells having transverse microfibrils on the inner surface of the wall were observed more frequently in GA3-treated epicotyls than in untreated or water-treated ones. This effect of GA3 was negated by simultaneously supplied colchicine. A crossed polylamellate structure was observed in the inner portion of the walls of GA3-treated cells, but not in the inner portion of the walls of colchicine-treated cells. The wall formed under the influence of colchicine consisted of microfibrils running in the same direction.

The change of pattern in microfibril arrangement on the inner surface of the cell wall of Closterium acerosum during cell growth.

Closterium acerosum (Schrank) Ehrenberg cells cultured on cycles of 16 h light and 8 h dark, undergo cell division synchronously in the dark period. After cell division, the symmetry of the daughter semicells is restored by controlled expansion, the time required for this restoration, 3.5-4 h, being relatively constant. The restoration of the symmetry is achieved by highly oriented surface expansion occurring along the entire length of the new semicell. During early semicell expansion, for about 2.5 h, microfibrils are deposited parallel to one another and transversely to the cell axis on the inner surface of the new wall. Wall microtubules running parallel to the transversely oriented microfibrils are observed during this period. About 2.5 h after septum formation, preceding the cessation of cell elongation, bundles of 7-11 microfibrils running in various directions begin to overlay the parallel-arranged microfibrils already deposited. In the fully elongated cells, no wall microtubules are observed.

Effects of colchicine on cell shape and on microfibril arrangement in the cell wall of Closterium acerosum.

Cell morphogenesis in Closterium acerosum (Schrank) Ehrenberg was greatly influenced by colchicine. Addition of colchicine to the medium led to production of "tadpole-shaped" cells, by decreasing the length and increasing the thickness of the new semicells. Transversely oriented wall microtubules and microfibrils, characteristic of normally elongating semicells, were not observed in colchicine-treated semicells, randomly oriented microfibrils being present instead. About 3.5 h after septum formation, the randomly oriented microfibrils began to be overlaid by bundles of microfibrils as seen in normal semicells at the later stage of elongation. When colchicine treatment was terminated 1 h after septum formation, cell elongation was partially restored and microfibrils were deposited parallel to each other and transversely to the cell axis, indicating that the effect of colchicine on microfibril arrangement in growing semicells is reversible.

Antagonisms between Kinetin and Amino Acids: Experiments on the Mode of Action of Cytokinins.

The maintenance of chlorophyll in darkened first leaves of oats was used as a bioassay for cytokinins in pea (Pisum sativum) roots. No cytokinin was found (in contrast with earlier reports on sunflower roots); however, the extracts contained two or more substances antagonistic to cytokinin, i. e., promoting the yellowing in this test. Because the most active of these appeared to be an amino acid, individual amino acids were examined for their ability to modify the greening reaction. As a result, l-serine was found to have these properties. It promotes yellowing whether the greening agent is kinetin, indoleacetic acid, or adenine; it is, therefore, not functioning as a specific cytokinin antagonist. Its action is due to promoting proteolysis. Its d-isomer is inactive. l-Arginine, which alone does not cause chlorophyll retention and only weakly inhibits proteolysis, strongly antagonizes the action of l-serine, and thus prevents the yellowing; this effect is specific, and the only other effective serine antagonist found, although much weaker, is l-threonine. The action of arginine is not due to its preventing serine uptake, but rather the action parallels the serine-arginine antagonism previously described for nitrate reductase induction. A novel interpretation of the effect of amino acids on this process is therefore put forward. In studies of the RNase in darkened oat leaves, serine was found to have no effect; however, kinetin strongly inhibits the normal rise in the level of RNase which occurs in the isolated leaf. Kinetin also maintains the integrity of the cell membranes. A variety of evidence leads to the conclusion that the primary action of kinetin on the leaf is to inhibit proteolysis, rather than to promote protein synthesis.