Actin-microtubule interactions in the algaNitella: analysis of the mechanism by which microtubule depolymerization potentiates cytochalasin's effects on streaming

Summary In the characean algaNitella, depolymerization of microtubules potentiates the inhibitory effects of cytochalasins on cytoplasmic streaming. Microtubule depolymerization lowers the cytochalasin B and D concentrations required to inhibit streaming, accelerates inhibition and delays streaming recovery. Because microtubule depolymerization does not significantly alter³H-cytochalasin B uptake and release, elevated intracellular cytochalasin concentrations are not the basis for potentiation. Instead, microtubule depolymerization causes actin to become more sensitive to cytochalasin. This increased sensitivity of actin is unlikely to be due to direct stabilization of actin by microtubules, however, because very few microtubules colocalize with the subcortical actin bundles that generate streaming. Furthermore, microtubule reassembly, but not recovery of former transverse alignment, is sufficient for restoring the normal cellular responses to cytochalasin D. We hypothesize that either tubulin or microtubule-associated proteins, released when microtubules depolymerize, interact with the actin cytoskeleton and sensitize it to cytochalasin.Abbreviations APW artificial pond water - Ca_c cytoplasraic free calcium concentration - DMSO dimethyl sulfoxide - MT microtubule-minus - MT+ microtubule-plus.     

Amiprophos-methyl (APM): A rapid,reversible, anti-microtuble agent for plant cell cultures

Summary In plant cell suspension cultures sensitive to the herbicide amiprophos-methyl (APM), 1 to 3 M APM completely depolymerized both cortical and mitotic microtubule (MT) arrays in 1 hour. In comparison, a 2 hour application of 3 mM colchicine had no effect on MT arrays. Recovery from APM treatment occurred as early as 5 minutes after removal of APM. Short, cortical MTs were visible in 3 hours and complete MT arrays were found within 22 hours after drug removal.Sensitivity to APM-induced MT depolymerization varied according to species but was increased or decreased by varying the mitotic rate in cultures. The results indicated APM sensitivity was related to lowered stability of MT arrays in rapidly cycling cells. APM treatment may help distinguish stabilized cortical MTs in elongating cells and nonstabilized cortical MTs in rapidly dividing cells.Abbreviations MT microtubule - APM amiprophos-methyl - DMSO dimethyl sulfoxide - PBS phosphate buffered saline     

Effects of colchicine and amiprophos-methyl on microfibril arrangement and cell shape inAdiantum protonemal cells

Summary 5 mM colchicine and 1 g/ml amiprophos-methyl, known antimicrotubule agents, were applied to fernAdiantum protonemata under red light. Both drugs caused microtubule disruption and subsequent apical swelling of protonemal cells after certain lag periods. While the lag periods for the onset of microtubule disruption after application of the two drugs were different (within 15 minutes in amiprophos-methyl, 1 hour in colchicine), the lag periods of apical swelling after microtubule disruption were nearly the same (approx. 70 minutes). The results suggest that the apical swelling is a consequence of microtubule disruption.In cells examined 1 hour after microtubule disruption by either drug, the microfibril arrangement of the innermost layer of the cell wall was random at the tip, transverse in the subapical region, and roughly longitudinal in the cylindrical region. This pattern of microfibrils was similar to that of untreated cells in which the microtubules show a similar arrangement (Murata and Wada 1989). Surprisingly, even after approx. 4 hours of microtubule disruption, when apical swelling had occurred in most cells, the pattern of microfibril deposition was not altered. The role of microtubules in oriented microfibril deposition and the mechanism of control of cell shape are discussed.Abbreviations APM amiprophos-methyl - DMSO dimethylsulfoxide - MT(s) microtubule(s) - PBS phosphate buffered saline     

A comparison of the effects of various spindle toxins on metaphase arrest and formation of micronuclei in cell-suspension cultures ofNicotiana plumbaginifolia

The effects of the spindle toxins colchicine, oryzalin and amiprophos-methyl (APM) on metaphase arrest, chromosome scattering, and on the induction and yield of micronuclei were compared in suspension cells ofNicotiana plumbaginifolia (kanamycin-resistant “Doba” line). The inhibition of spindle formation is stronger with oryzalin and APM than with colchicine, which resulted in a more efficient accumulation of meta-phases with well-scattered chromosomes, allowing the isolation of single chromosomes. Further, APM and oryzalin treatments resulted in a higher frequency of micro-nucleated cells and greater yield of micronuclei than after colchicine treatment. The different actions of the chemicals on the functioning of the spindle, development of nuclear membranes around the chromosomes, formation of micronuclei and fusion of micronuclei, resulting in restitution nuclei, are discussed.     

The organization of F-actin in root tip cells ofAdiantum capillus veneris throughout the cell cycle

Summary The patterns of F-actin in relation to microtubule (Mt) organization in dividing root tip cells ofAdiantum capillus veneris were studied with rhodamine-phalloidin (RP) labelling and tubulin immunofluorescence. Interphase cells display a well organized network of cortical/subcortical, endoplasmic and perinuclear actin filaments (AFs), not particularly related to the interphase Mt arrays. The cortical AFs seem to persist during the cell cycle while the large subcortical AF bundles disappear by preprophase/prophase and reappear after cytokinesis is completed. In some but not all of the preprophase cells the cortical AFs tend to form a band (AF-PPB) coincident with the preprophase band of Mts (Mt-PPB). In metaphase and anaphase cells AFs are localized in the cell cortex, around the spindle and inside it coincidently with kinetochore Mt bundles. During cytokinesis AFs are consistently found in the phragmoplast. In oryzalin treated cells neither Mt-PPBs, spindles and phragmoplasts exist, nor such F-actin structures can be observed. In cells recovering from oryzalin, AF-PPBs, AF kinetochore bundles and AF phragmoplasts reform. They show the same pattern with the reinstating respective Mt arrays. In contrast, in cells treated with cytochalasin B (CB), AFs disappear but all categories of Mt arrays form normally.These observations show that F-actin organization in root tip cells ofA. capillus veneris differs from that of root tip cells of flowering plants examined so far. In addition, Mts seem to be crucial for F-actin organization as far as it concerns the PPB, the mitotic spindle, and the phragmoplast.Abbreviations AF actin filament - CB cytochalasin B - MBS m-male-imidobenzoyl-N-hydroxysuccinimide ester - MSB microtubule stabilizing buffer - Mt microtubule - PBS phosphate buffered saline - PPB preprophase band - RP rhodamine phalloidin     

Probing microtubule organizing centres with MPM-2 in dividing cells of higher plants using immunofluorescence and immunogold techniques

Summary The localization in higher plant cells of phosphorylated proteins recognized by the monoclonal antibody MPM-2 was investigated, with particular attention to putative microtubule organizing centres (MTOCs). Immunofluorescence and immunogold electron microscopy showed that MPM-2 did not localize with most putative MTOCs in cells and protoplasts of the gymnospermPicea glauca and in cells of the angiospermVicia faba. The distribution of phosphoproteins detected by MPM-2 was similar during mitosis in both species. At late interphase and early prophase MPM-2 preferentially labelled nucleoli and the region around the condensing chromosomes but not the cytoplasm. General labelling of the cytoplasm followed dissolution of the nuclear envelope and by prometaphase centromeres stained strongly. At metaphase and very early anaphase kinetochores stained strongly by immunofluorescence but only weakly using immunogold; spindle microtubules (MTs) showed little staining. Kinetochore staining disappeared during anaphase and by telophase centromeres and loose regions of chromatin in reforming nuclei were labelled. Treatment with the anti-microtubular drug amiprophosmethyl (APM) showed that the phosphorylation/dephosphorylation cycle detected by MPM-2 proceeded independently of the mitotic spindle. Staining of centromeres/kinetochores with MPM-2 suggests that phosphorylation and dephosphorylation of this region of mitotic chromosomes may be involved in chromosome organization, chromatid separation and MT nucleation and/or attachment.Abbreviations APM amiprophos-methyl - DAPI 4,6-diamidino-2-phenylindole - EGTA ethylene glycol-bis(-aminoethyl ether) - FITC fluorescein isothiocyanate - MT microtubule - MTOC microtubule organizing centre - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline - PBSB phosphate buffered saline with bovine serum albumin - PIPES piperazine-N,N-bis (2-ethanesulfonic acid) - PPB preprophase band - SPB spindle pole body - TRITC tetramethylrhodamine isothiocyanate     

Microtubule distribution in gravitropic protonemata of the mossCeratodon

Summary Tip cells of dark-grown protonemata of the mossCeratodon purpureus are negatively gravitropic (grow upward). They possess a unique longitudinal zonation: (1) a tip group of amylochloroplasts in the apical dome, (2) a plastid-free zone, (3) a zone of significant plastid sedimentation, and (4) a zone of mostly non-sedimenting plastids. Immunofluorescence of vertical cells showed microtubules distributed throughout the cytoplasm in a mostly axial orientation extending through all zones. Optical sectioning revealed a close spatial association between microtubules and plastids. A majority (two thirds) of protonemata gravistimulated for >20 min had a higher density of microtubules near the lower flank compared to the upper flank in the plastid-free zone. This apparent enrichment of microtubules occurred just proximal to sedimented plastids and near the part of the tip that presumably elongates more to produce curvature. Fewer than 5% of gravistimulated protonemata had an enrichment in microtubules near the upper flank, whereas 14% of vertical protonemata were enriched near one of the side walls. Oryzalin and amiprophos-methyl (APM) disrupted microtubules, gravitropism, and normal tip growth and zonation, but did not prevent plastid sedimentation. We hypothesize that a microtubule redistribution plays a role in gravitropism in this protonema. This appears to be the first report of an effect of gravity on microtubule distribution in plants.Abbreviations APM amiprophos-methyl - DIC differential interference contrast - DMSO dimethyl sulfoxide - EGTA ethylene glycolbis-(-amino-ethylether) N,N,N',N'-tetraacetic acid - FITC fluorescein isothiocyanate - GS gravitropic stimulus - MT microtubule - PIPES piperazine-N,N'-bis-2-ethanesulfonic acid     

Structure and assembly of the cortical microtubule cytoskeleton in the green alga,Boodlea coacta

Summary Examination was made of the structure and assembly of the cortical microtubule (MT) cytoskeleton in the coenocytic green algaBoodlea coacta (Dickie) Murray et De Toni by immunofluorescence microscopy. Cortical MTs inBoodlea protoplasts are arranged randomly but some show a meridional arrangement within 6 h after protoplast formation. At 6–9 h such MTs become highly concentrated and parallel to each other in certain areas. At 12 h the concentration is uniformly high throughout the cell, indicating the completion of high density meridional arrangement of cortical MTs. Cortical MTs exhibiting a high density, meridional arrangement show characteristic disassembly by treatment with 10 M amiprophos-methyl (APM) or cold treatment (0 °C). Disassembly occurs by each MT unit at positions skipping 30–40 m in the transverse direction, and neighboring MTs subsequently disassemble to form MT groups. Each group becomes slender and then disappears completely within the following 24 h. The meridional arrangement of cortical MTs is disrupted by N-ethylmaleimide (NEM) accompanied by a remarkable reduction in density. The remaining MTs form groups at 30–40 m intervals from each other, as also occurs with drug or cold treatment, but disruption and density return to normal levels following removal of NEM. It appears that there are meridionally oriented channels, anchor-rich and anchor-poor, in the plasma membrane. The channels could be distributed alternately and anchors could be deposited in a cross-linking manner with cortical MTs to form a stable cortical MT-cytoskeleton. MTs comprising the cortical MT cytoskeleton could be oriented by meridionally oriented channels of anchors which are distributed following establishment of cell polarity.Abbreviations APM amiprophos-methyl - MT microtubule - MTOC microtubule organizing center - NEM N-ethylrnaleimide     

Possible involvement of membrane fluidity in helicoidal microfibrillar orientation in the coenocytic green alga,Boergesenia forbesii

Summary Microfibrillar textures and orientation of cellulose microfibrils (MFs) in the coenocytic green alga,Boergesenia forbesii, were investigated by fluorescence and electron microscopy. Newly formed aplanosporic spherical cells inBoergesenia start to form cellulose MFs on their surfaces after 2 h of culture at 25°C. Microfibrillar orientation becomes random, fountain-shaped, and helicoidal after 2, 4, and 5 h, respectively. The fountain orientation of MFs is usually apparent prior to helicoidal MF orientation and thus may be considered to initiate helicoid formation. Microfibrils continue to take on the helicoidal arrangement during the growth ofBoergesenia thallus. The helicoidal orientation of MFs occurs through gradual counterclockwise change in MF deposition by terminal complexes (TCs) viewed from inside the cell. On the dorsal side of curving TC impressions in helicoidal texture formation on a freeze-fractured plasma membrane, the aggregation of intramembranous particles (IMPs) occurs. Membrane flow may thus possibly affect the regulation of helicoidal orientation inBoergesenia. Following treatment with 3 M amiprophos-methyl (APM) or 1 mM colchicine, cortical microtubules (MTs) completely disappear within 24 h but helicoidal textures formation is not affected. With 15 M cytochalasin B or 30 M phalloidin, however, the helicoidal orientation of MFs becomes random. Treatment with CaCl₂ (10 mM) causes the helicoidal MF orientation of cells to become random, but co-treatment with N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) (100 mM) prevents this effect, though W-7 has no effect on the helicoidal MF formation. It thus follows that MF orientation inBoergesenia possibly involves actin whose action may be regulated by calmodulin.Abbreviations APM amiprophos-methyl - DMSO dimethylsulfoxide - IMP intramembranous particle - MF microfibril - MT microtubule - TC terminal complex; W-7 N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide     

Mitotic disrupter herbicides act by a single mechanism but vary in efficacy

Summary Although there are numerous herbicides that disrupt mitosis as a mechanism of action, to date not one has compared the effects of these disrupters on a single species and over a range of concentrations. Oat seedlings, treated with a range of concentrations of nine different mitotic disrupter herbicides, were examined by immunofluorescence microscopy of tubulin in methacrylate sections. All herbicides caused the same kinds of microtubule disruption, although the concentrations required to cause the effects differed markedly between the herbicides. Effects on spindle and phragmoplast mitotic microtubule arrays were seen at the lowest concentrations and manifested as multipolar spindles and bifurcated phragmoplasts (which subsequently resulted in abnormal cell plate formation). At increasing concentrations, effects on mitotic microtubule arrays manifested as microtubule tufts at kinetochores and reduction of cortical microtubules resulting in arrested prometaphase figures and isodiametric cells. These data indicate that all mitotic disrupter herbicides have a common primary mechanism of action, inhibition of microtubule polymerization, and that marginal effects observed in the past were the result of incomplete inhibition and/or differential sensitivity of the microtubule arrays.Abbreviations DCPA 2,3,5,6-tetrachloroterephthalic acid dimethyl ester - APM amiprophosmethyl - DAPI 4,6-diamidino-2-phenyl indole - MTOC microtubule organizing center     

Plasma membrane-cell wall connections: Roles in mitosis and cytokinesis revealed by plasmolysis ofTradescantia virginiana leaf epidermal cells

Summary Tradescantia virginiana leaf epidermal cells were plasmolysed by sequential treatment with 0.8 M and 0.3 M sucrose. Plasmolysis revealed adhesion of the plasma membrane to the cell wall at sites coinciding with cytoskeletal arrays involved in the polarisation of cells undergoing asymmetric divisions — cortical actin patch — and in the establishment and maintenance of the division site —preprophase band of microtubules and filamentous (F) actin. The majority of cells retained adhesions at the actin patch throughout mitosis. However, only approximately 13% of cells formed or retained attachments at the site of the preprophase band. After the breakdown of the nuclear envelope, plasmolysis had a dramatic effect on spindle orientation, cell plate formation, and the plane of cytokinesis. Spindles were rotated at abnormal angles including tilted into the plane of the epidermis. Cell plates formed but were quickly replaced by vacuole-like intercellular compartments containing no Tinopal-stainable cell wall material. This compartment usually opened to the apoplast at one side, and cytokinesis was completed by the furrow extending across the protoplast. This atypical cytokinesis was facilitated by a phragmoplast containing microtubules and F-actin. Progression of the furrow was unaffected by 25 g of cytochalasin B per ml but inhibited by 10 M oryzalin. Phragmoplasts were contorted and misguided and cytokinesis prolonged, indicating severe disruption to the guidance mechanisms controlling phragmoplast expansion. These results are discussed in terms of cytoskeleton-plasma membrane-cell wall connections that could be important to the localisation of plasma membrane molecules defining the cortical division site and hence providing positional information to the cytokinetic apparatus, and/or for providing an anchor for cytoplasmic F-actin necessary to generate tension on the phragmoplast and facilitate its directed, planar expansion.Abbreviations ADZ actin-depleted zone - DIC differential interference contrast - GMC guard mother cell - MT microtubule - PPB preprophase band - SMC subsidiary mother cell Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Competitive Inhibition of High-Affinity Oryzalin Binding to Plant Tubulin by the Phosphoric Amide Herbicide Amiprophos-Methyl

Amiprophos-methyl (APM), a phosphoric amide herbicide, was previously reported to inhibit the in vitro polymerization of isolated plant tubulin (L.C. Morejohn, D.E. Fosket [1984] Science 224: 874-876), yet little other biochemical information exists concerning this compound. To characterize further the mechanism of action of APM, its interactions with tubulin and microtubules purified from cultured cells of tobacco (Nicotiana tabacum cv Bright Yellow-2) were investigated. Low micromolar concentrations of APM depolymerized preformed, taxol-stabilized tobacco microtubules. Remarkably, at the lowest APM concentration examined, many short microtubules were redistributed into fewer but 2.7-fold longer microtubules without a substantial decrease in total polymer mass, a result consistent with an end-to-end annealing of microtubules with enhanced kinetic properties. Quasi-equilibrium binding measurements showed that tobacco tubulin binds [14C]oryzalin with high affinity to produce a tubulin-oryzalin complex having a dissociation constant (Kd) = 117 nM (pH 6.9; 23[deg]C). Also, an estimated maximum molar binding stoichiometry of 0.32 indicates pharamacological heterogeneity of tobacco dimers and may be related to structural heterogeneity of tobacco tubulin subunits. APM inhibits competitively the binding of [14C]oryzalin to tubulin with an inhibition constant (Ki) = 5 [mu]M, indicating the formation of a moderate affinity tubulin-APM complex that may interact with the ends of microtubules. APM concentrations inhibiting tobacco cell growth were within the threshold range of APM concentrations that depolymerized cellular microtubules, indicating that growth inhibition is caused by microtubules depolymerization. APM had no apparent effect on microtubules in mouse 3T3 fibroblasts. Because cellular microtubules were depolymerized at APM and oryzalin concentrations below their respective Ki and Kd values, both herbicides are proposed to depolymerize microtubules by a substoichiometric endwise mechanism.     

Vascular differentiation in the root cortex of peas: Premitotic stages of cytoplasmic reactivation

Summary The spatial and temporal organization of the microtubular cytoskeleton at the transitional stage of mitosis and cytokinesis has been studied in the chaetophoralean green algaAphanochaete magna using indirect immunofluorescence light microscopy and transmission electron microscopic analysis of serial sections including computer-aided three-dimensional reconstruction. At late mitosis, elaborate asterlike microtubule systems including bundles interconnecting both centriolar regions are present. These systems disappear a the onset of interzonal spindle disintegration. The incipient phycoplast consists of a star-shaped microtubule assemblage projecting from the intact interzonal spindle. It develops strongly at the time of spindle disintegration, later on it becomes compressed by daughter nuclei movement. Cell plate formation is associated with a two-dimensional phycoplast. Phycoplast microtubules remain for a while associated with the completed cross wall but finally they depolymerize. The general occurrence of astral microtubule systems (includingA. magna) is evaluated. The subsequent developmental stages of the phycoplast, formation, maturation and depolymerization, are discussed.Abbreviations IF immunofluorescence - IZS interzonal spindle - MT microtubule - MTOC microtubule organizing center - TEM transmission electron microscopy     

Association of γ‐tubulin with kinetochore/centromeric region of plant chromosomes

Monoclonal antibodies raised against a phylogenetically conserved peptide from the C-terminal domain of γ-tubulin molecule were used for immunofluorescence detection of γ-tubulin in acentriolar mitotic spindles of plant cells. The antibodies stained kinetochore fibres along their whole length, including the close vicinity of kinetochores. After microtubule disassembly by the antimicrotubular drugs amiprophos-methyl, oryzalin and colchicine, γ-tubulin was found on remnants of kinetochore fibres attached to chromosomes. In cells recovering from the amiprophos-methyl treatment, γ-tubulin was localized with the re-growing kinetochore microtubule fibres nucleated or captured by kinetochore/centromeric regions. On isolated chromosomes, γ-tubulin co-localized with α-tubulin in the kinetochore/centromeric region. The data presented suggest that in acentriolar higher plant cells γ-tubulin might be directly or indirectly involved in modulation and/or stabilization of kinetochore–microtubule interactions.     

Effects of Acetylcholine, Cytochalasin B and Amiprophos methyl on Phloem Transport in Radish (Raphanus sativas)

We investigated the role of the "sieve tube-companion cell complex" lining the tube periphery, particularly the microfilament and microtubule, in assisting the pushing of phloem sap flow. We made a simple phloem transport system with a living radish plant, in which the conducting channel was exposed for local treatment with chemicals that are effective in modulating protoplasmic movement (acetylcholine, (ACh) a neurotransmitter in animals and insects; cytochalasin B, (CB) a specific inhibitor of many cellular responses that are mediated by microfilament systems and amiprophos-methyl, (APM) a specific inhibitor of many cellular responses that are mediated by microtubule systems). Their effects on phloem transport were estimated by two experimental devices: (i) a comparison of changes in the amount of assimilates in terms of carbohydrates and 14C-labeled photosynthetic production that is left in the leaf blade of treated plants; and (ii) distribution patterns of 14C-labeled leaf assimilates in the phloem transport system. The results indicate that CB and APM markedly inhibited the transfer of photosynthetic product from leaf to root via the leaf vein, while ACh enhanced the transfer of photosynthetic product in low concentrations (5.0×10-4 mol/L) but inhibited it in higher concentrations (2.0×10-3 mol/L) from leaf to root via the leaf vein. Autoradiograph imaging clearly reveals that ACh treatment is more effective than the control, and both CB and APM treatments effectively inhibit the passage of radioactive assimilates. All of the results support the postulation that the peripheral protoplasm in the sieve tube serves not only as a passive semi-permeable membrane, but is also directly involved in phloem transport.     

Role of the microtubule cytoskeleton in alternating changes in cellulose-microfibril orientation in the coenocytic green alga,Chaetomorpha moniligera

The functions of the microtubule (MT) cytoskeleton in changing the orientation of microfibrils (MFs) in the cell walls of the coenocytic green alga Chaetomorpha moniligera Kjellman were investigated by electron microscopy. The cortical MT cytoskeleton in Chaetomorpha was comprised of longitudinally oriented MTs. Cellulose MFs, however, alternately changed their orientation longitudinally and transversely to form crisscross MF textures. Microtubules were parallel to longitudinally oriented MFs but never to those that were transversely oriented. The average density of MTs during the formation of longitudinally oriented MFs was 216 per 50 m of wall and that of transversely oriented MFs 170/50 m. To determine exactly the MT-density dependency of each MF orientation, changes in MF orientation were examined by changing MT density after treating and removing amiprophos-methyl (APM). Microtubules were reduced in number by a half (100/50 m) after 2 h and by 3/4 (50/50 m) after 3 h of treatment with APM (3 mM). This reduction was caused by the disappearance of alternating MTs. Microtubules retained this density (50/ 50 m) up to 6 h, and then gradually disappeared within 24 h. Microfibril orientation in the innermost cell wall was transverse after treatment with APM for 2 h but was helicoidal after 6 h. Polymerization of MTs occurred in the longitudinal direction following the removal of APM after treatment for 48 h. Microtubule density rose to about 100/50 m and 200/50 m after 6 h and 24 h, respectively. The orientation of MTs changed from helicoidal to transverse and transverse to longitudinal after 6 h and 24 h, respectively. When APM was removed prior to formation of the helicoidal texture, longitudinally oriented MFs appeared within 6 h. There is thus an alternating cycle of formation of longitudinally and transversely oriented MFs within a 12-h period. Formation of transversely oriented MFs as a result of APM treatment started in the middle of a cell as hoops which then extended in the apical and basal directions. Formation of longitudinally oriented MFs as a result of the removal of APM started from the apical end and proceeded toward the base. It follows from these results that: (1) the point of formation of longitudinally oriented MFs differs from that for transversely oriented MFs, (2) MF orientation in each case depends on a separately functioning mechanism, (3) MT density changes rhythmically to trigger a switch for crisscross orientation of MFs.Abbreviations APM amiprophos-methyl - MF microfibril - MT microtubule - TC terminal complex We thank Dr. K. Okuda for making helpful discussion and Miss. T. Matsuki for assistance with replica preparation.     

Microtubules do not control orientation of secondary cell wall microfibril deposition inMicrasterias

Summary The influence of the microtubule disorganizing substances amiprophos-methyl (APM) and colchicine on secondary wall formation inMicrasterias denticulata was investigated by the freezeetch technique. The results reveal that neither microtubule inhibitor changes the pattern of microfibril deposition. The application of APM or colchicine also does not cause any structural alterations of the microfibrils or of the protoplasmic (Pf) and the exoplasmic (Ef) fracture face of the plasma membrane, thus indicating that microtubules are not involved in secondary wall formation inM. denticulata. However, since areas of the plasma membrane which collapsed upon freeze-etching are restricted to the Pf-face of cells treated with microtubule inhibitors, cortical microtubules may function as mechanical support during secondary wall formation. In the cortical cytoplasm filamentous structures are found in close spatial relationship and an almost parallel alignment to rosettes of the plasma membrane.     

Mutants Resistant to anti-Microtubule Herbicides Map to a Locus on the Uni Linkage Group in Chlamydomonas Reinhardtii

We have used genetic analysis to study the mode of action of two anti-microtubule herbicides, amiprophos-methyl (APM) and oryzalin (ORY). Over 200 resistant mutants were selected by growth on APM- or ORY-containing plates. The 21 independently isolated mutants examined in this study are 3- to 8-fold resistant to APM and are strongly cross-resistant to ORY and butamiphos, a close analog of APM. Two Mendelian genes, apm1 and apm2, are defined by linkage and complementation analysis. There are 20 alleles of apm1 and one temperature-sensitive lethal (33°) allele of apm2. Mapping by two-factor crosses places apm1 6.5 cM centromere proximal to uni1 and within 4 cM of pf7 on the uni linkage group, a genetically circular linkage group comprising genes which affect flagellar assembly or function; apm2 maps near the centromere of linkage group VIII. Allele-specific synthetic lethality is observed in crosses between apm2 and alleles of apm1. Also, self crosses of apm2 are zygotic lethal, whereas crosses of nine apm1 alleles inter se result in normal germination and tetrad viability. The mutants are recessive to their wild-type alleles but doubly heterozygous diploids (apm1 +/+ apm2) made with apm2 and any of 15 apm1 alleles display partial intergenic noncomplementation, expressed as intermediate resistance. Diploids homozygous for mutant alleles of apm1 are 4-6-fold resistant to APM and ORY; diploids homozygous for apm2 are ts(-) and 2-fold resistant to the herbicides. Doubly heterozygous diploids complement the ts(-) phenotype of apm2, but they are typically 1.5-2-fold resistant to APM and ORY. From the results described we suggest that the gene products of apm1 and apm2 may interact directly or function in the same structure or process.     

Microtubule cytoskeleton in intact and wounded coenocytic green algae

Microtubule (MT) arrangements were investigated, with immunofluorescence and electron microscopy, in two related species of coenocytic green algae. Intact cells of both Ernodesmis verticillata (Kützing) Boergesen and Boergesenia forbesii (Harvey) Feldmann have two morphologically distinct populations of MTs: a highly regular cortical array consisting of a single layer of parallel, longitudinal MTs; and perinuclear MTs radiating from the surface of the envelope of each interphase nucleus. In both algae, mitotic figures lack perinuclear MTs around them. Pre-incubation with taxol does not alter the appearance of these arrays. The cortical and nuclear MTs appear to coexist throughout the nuclear cycle, unlike the condition in most plant cells. At the cut/contracting ends of wounded Ernodesmis cells, cortical MTs exhibit bundling and marked convolution, with some curvature and slight bundling of MTs throughout the cell cortices. In Boergesenia, wound-induced reticulation and separation of the protoplasm into numerous spheres also involves a fasciation of MTs within the attenuating regions of the cytoplasm. Although some cortical MTs are fairly resistant to cold and amiprophos-methyl-induced depolymerization, the perinuclear ones are very labile, depolymerizing in 5–10 min in the cold. The MT cytoskeleton is not believed to be directly involved in wound-induced motility in these plants because amiprophos-methyl and cold depolymerize most cortical MTs without inhibiting motility. Also, the identical MT distributions in intact cells of these two algae belie the very different patterns of cytoplasmic motility. Although certain roles of the MT arrays may be ruled out, their exact functions in these plants are not known.Abbreviations APM amiprophos-methyl - DIC differential interference contrast - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MT(s) microtubule(s) - PBS phosphate-buffered saline     

Phosphatidic acid integrates calcium signaling and microtubule dynamics into regulating ABA-induced stomatal closure in Arabidopsis

Specific cellular components have been identified to function in abscisic acid (ABA) regulation of stomatal apertures, including calcium, the cytoskeleton, and phosphatidic acid. In this study, the regulation and dynamic organization of microtubules during ABA-induced stomatal closure by phospholipase D (PLD) and its product PA were investigated. ABA induced microtubule depolymerization and stomatal closure in wide-type (WT) Arabidopsis, whereas these processes were impaired in PLD mutant (pldα1). The microtubule-disrupting drugs oryzalin or propyzamide induced microtubule depolymerization, but did not affect the stomatal aperture, whereas their co-treatment with ABA resulted in stomatal closure in both WT and pldα1. In contrast, the microtubule-stabilizing drug paclitaxel arrested ABA-induced microtubule depolymerization and inhibited ABA-induced stomatal closure in both WT and pldα1. In pldα1, ABA-induced cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) elevation was partially blocked, and exogenous Ca²⁺-induced microtubule depolymerization and stomatal closure were impaired. These results suggested that PLDα1 and PA regulate microtubular organization and Ca²⁺ increases during ABA-induced stomatal closing and that crosstalk among signaling lipid, Ca²⁺, and microtubules are essential for ABA signaling.