The motility of Chara corallina myosin was inhibited reversibly by 2,3-butanedione monoxime (BDM)

We studied the effects of 2,3-butanedione monoxime (BDM) on the cytoplasmic streaming of Chara corallina and on the motility of myosin prepared from the same plant to examine whether this reagent really affects the plant class XI myosin. It was found that BDM inhibited both cytoplasmic streaming and the motility of myosin at a very similar concentration range (10-100 mM). BDM introduced directly into tonoplast-free cells also inhibited cytoplasmic streaming. These results suggested that effect of BDM on cytoplasmic streaming was exerted through myosin and not through ion channels at least in Chara corallina, though a very high concentration of BDM was required.     

Is 2,3-butanedione monoxime an effective inhibitor of myosin-based activities in plant cells?

Summary The effectiveness of 2,3-butanedione monoxime (BDM) as an inhibitor of plant myosins has been investigated. Three myosin-dependent motility phenomena in plants, namely cytoplasmic streaming inChara corallina, light-dependent chloroplast repositioning inElodea sp., and brefeldin A(BFA)-induced Golgi membrane dynamics in wheat (Triticum aestivum L. cv. Kite) roottip cells were investigated. All three processes were inhibited by the sulfhydryl-modifying agent N-ethylmalemide (NEM), indicating the probable involvement of myosin as the motor protein in each case. However, none of these myosin-dependent processes were inhibited by BDM at concentrations as high as 20 mM in some instances. These results therefore question the general usefulness of BDM as an inhibitor of myosin-based activities in plant cells.     

Actin cytoskeleton is responsible for the change of cytoplasmic organization in root hair cells induced by a protein phosphatase inhibitor, calyculin A

Summary In root hair cells ofLimnobium stoloniferum, a protein phosphatase inhibitor, calyculin A (CA), at concentrations higher than 50 nM inhibits cytoplasmic streaming and induces remarkable morphological changes in the cytoplasm: the transvacuolar strands disperse and spherical cytoplasmic bodies emerge. The mechanism of the morphological changes of the cytoplasm induced by CA was studied by pharmacological analyses. The formation of spherical bodies in cells treated with CA was suppressed by the actin-depolymerizing and -fragmenting drugs latrunculin B and cytochalasin D at concentrations higher than 100 nM and 5 M, respectively. In contrast, 100 M propyzamide, a microtubule-depolymerizing drug, did not affect the formation of spherical bodies by CA. Interestingly, 60 mM 2,3-butanedione monoxime, an inhibitor of myosin, also suppressed the CA-induced formation of cytoplasmic spherical bodies. These results indicate that the actin cytoskeleton is intimately involved in the morphological changes of the cytoplasm induced by CA.Abbreviations APW artificial pond water - BDM 2,3-butanedione monoxime - CD cytochalasin D - DMSO dimethylsulfoxide - LB latrunculin B - Pro propyzamide     

Is 2,3-butanedione monoxime an effective inhibitor of myosin-based activities in plant cells?

The effectiveness of 2,3-butanedione monoxime (BDM) as an inhibitor of plant myosins has been investigated. Three myosin-dependent motility phenomena in plants, namely cytoplasmic streaming in Chara corallina, light-dependent chloroplast repositioning in Elodea sp., and brefeldin A(BFA)-induced Golgi membrane dynamics in wheat (Triticum aestivum L. cv. Kite) root-tip cells were investigated. All three processes were inhibited by the sulfhydryl-modifying agent N-ethylmalemide (NEM), indicating the probable involvement of myosin as the motor protein in each case. However, none of these myosin-dependent processes were inhibited by BDM at concentrations as high as 20 mM in some instances. These results therefore question the general usefulness of BDM as an inhibitor of myosin-based activities in plant cells.     

2,3-Butanedione monoxime increases sensitivity to Nikkomycin Z in the budding yeast Saccharomyces cerevisiae

Summary Nikkomycin Z (NZ) is a competitive inhibitor of chitin synthase III in the yeast Saccharomyces cerevisiae. Myosin type II-deficient yeast strains (myo1) display a dramatic reduction in growth when chitin synthase III activity is inhibited by NZ, supporting the contention that actomyosin motility plays an important role in maintaining cell wall integrity. A proposed inhibitor of cortical actin polymerization in vitro, 2,3-butanedione monoxime (BDM), also inhibits growth of wild-type yeast strains at a concentration of 20 mM. In this study, we assayed for potential in vivo interplay between BDM-sensitive cell functions and cell wall chitin synthesis by testing for increased sensitivity to NZ during co-treatment with BDM at sub-inhibitory concentrations. Our results show that BDM can increase the sensitivity of yeast cells to Nikkomycin Z.     

Aluminium causes variable responses in actin filament cytoskeleton of the root tip cells of Triticum turgidum

Summary. The effects of aluminium on the actin filament (AF) cytoskeleton of Triticum turgidum meristematic root tip cells were examined. In short treatments (up to 2 h) with 50–1000 μM AlCl₃·6H₂O, interphase cells displayed numerous AFs arrayed in thick bundles that lined the plasmalemma and traversed the endoplasm in different directions. Measurements using digital image analysis and assessment of the overall AF fluorescence revealed that, in short treatments, the affected cells possessed 25–30% more AFs than the untreated ones. The thick AF bundles were not formed in the Al-treated cells in the presence of the myosin inhibitors 2,3-butanedione monoxime (BDM) and 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7), a fact suggesting that myosins are involved in AF bundling. In longer Al treatments, the AF bundles were disorganised, forming granular actin accumulations, a process that was completed after 4 h of treatment. In the Al-treated cells, increased amounts of callose were uniformly deposited along the whole surface of the cell walls. In contrast, callose formed local deposits in the Al-treated cells in the presence of cytochalasin B, BDM, or ML-7. These results favour the hypothesis that the actomyosin system in the Al-treated cells, among other roles, participates in the mechanism controlling callose deposition. Correspondence and reprints: Department of Botany, Faculty of Biology, University of Athens, Athens 157 84, Greece.     

Actomyosin promotes cell plate alignment and late lateral expansion in <Emphasis Type="Italic">Tradescantia</Emphasis> stamen hair cells

Cytokinesis in higher-plant cells involves the formation of a cell plate in the interzone between the separating chromatids. The process is directed by the phragmoplast, an array of microtubules, actin filaments, and membranous elements. To determine if the role of actin in cytokinesis is dependent on myosin, we treated stamen hair cells of Tradescantia virginiana L. with 2,3-butanedione monoxime (BDM), an inhibitor of myosin ATPase and ML-7, a specific inhibitor of myosin light-chain kinase. Treatment with BDM resulted in a tilted cytokinetic apparatus during early initiation and a wavy cell plate with curved phragmoplasts during late lateral expansion. Treatment with ML-7 also resulted in inefficient late lateral expansion of the cell plate, with effects ranging from slower expansion to complete inhibition. Taken together, these results implicate myosin in the control of cell plate expansion and alignment.     

Localization of a 170 kDa myosin heavy chain in plant cells

Summary A polyclonal antibody directed against a 170 kDa myosin heavy chain from lily pollen tubes was employed to (a) assess the cellular distribution of the polypeptide using immunofluorescence methods, and (b) ascertain if similar polypeptides are present in pollen tubes and somatic cells of other species. Fluorescence is associated with particles of various size as well as an amorphous component, and is concentrated in the apical cytoplasm of lily and tobacco pollen tubes. Apical fluorescence is more extensive in lily than in tobacco, which may be related to different streaming patterns and apical zonation seen at the ultrastructural level. In suspension cells of tobacco andArabidopsis, fluorescence is concentrated around the nuclei. Dual localizations indicate that anti-myosin fluorescence may be associated with the presence of actin. Little or no staining was seen in controls consisting of either pre-immune serum or mono-specific IgG that had been preadsorbed with the 170 kDa polypeptide. Immunoblots show that a 170 kDa immunoreactive polypeptide is present in pollen tubes of tobacco andTradescantia virginiana in addition to lily, and in suspension culture cells of tobacco andArabidopsis and extracts of wholeArabidopsis seedlings. Our results show that a conserved 170 kDa myosin heavy chain is present in a variety of monocot and dicot cells. They are also consistent with the presence of multiple myosins in plants in general and pollen tubes in particular.Abbreviations BSA bovine serum albumin - IgG immunoglobulin G - Mf microfilament - Mt microtubule - PBS phosphate-buffered saline - PME 50 mM Pipes, 5mM EGTA - 2mM MgSO₄, pH6.9.     

Inhibitors of myosin,but not actin,alter transport through <Emphasis Type="Italic">Tradescantia</Emphasis> plasmodesmata

Actin and myosin are components of plasmodesmata, the cytoplasmic channels between plant cells, but their role in regulating these channels is unclear. Here, we investigated the role of myosin in regulating plasmodesmata in a well-studied, simple system comprising single filaments of cells which form stamen hairs in Tradescantia virginiana flowers. Effects of myosin inhibitors were assessed by analysing cell-to-cell movement of fluorescent tracers microinjected into treated cells. Incubation in the myosin inhibitor, 2,3-butanedione monoxime (BDM) or injection of anti-myosin antibodies increased cell–cell transport of fluorescent dextrans, while treatment with the myosin inhibitor N-ethylmaleimide (NEM) decreased cell–cell transport. Pretreatment with the callose synthesis inhibitor, deoxy-d-glucose (DDG), enhanced transport induced by BDM treatment or injection of myosin antibodies but did not relieve NEM-induced reduction in transport. In contrast to the myosin inhibitors, cell-to-cell transport was unaffected by treatment with the actin polymerisation inhibitor, latrunculin B, after controlling for callose synthesis with DDG. Transport was increased following azide treatment, and reduced after injection of ATP, as in previous studies. We propose that myosin detachment from actin, induced by BDM, opens T. virginiana plasmodesmata whereas the firm attachment of myosin to actin, promoted by NEM, closes them.     

Muscle cross-bridges bound to actin are disordered in the presence of 2,3-butanedione monoxime.

Electron paramagnetic resonance spectroscopy was used to monitor the orientation of muscle cross-bridges attached to actin in a low force and high stiffness state that may occur before force generation in the actomyosin cycle of interactions. 2,3-butanedione monoxime (BDM) has been shown to act as an uncompetitive inhibitor of the myosin ATPase that stabilizes a myosin.ADP.P(i) complex. Such a complex is thought to attach to actin at the beginning of the powerstroke. Addition of 25 mM BDM decreases tension by 90%, although stiffness remains high, 40-50% of control, showing that cross-bridges are attached to actin but generate little or no force. Active cross-bridge orientation was monitored via electron paramagnetic resonance spectroscopy of a maleimide spin probe rigidly attached to cys-707 (SH-1) on the myosin head. A new labeling procedure was used that showed improved specificity of labeling. In 25 mM BDM, the probes have an almost isotropic angular distribution, indicating that cross-bridges are highly disordered. We conclude that in the pre-powerstroke state stabilized by BDM, cross-bridges are attached to actin, generating little force, with a large portion of the catalytic domain of the myosin heads disordered.