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.     

Identification of calmodulin in the green alga Mougeotia and its possible function in chloroplast reorientational movement

A soluble protein was isolated from Mougeotia by chloropromazine-sepharose 4 B affinity chromatography. The protein matches the properties of calmodulin in terms of heat stability, Ca²⁺-dependent electrophoretic mobility in sodium-dodecyl-sulfate polyacrylamide gels, and its ability to activate cyclic nucleotide phosphodiesterase in a Ca²⁺-dependent manner. Phytochrome-mediated chloroplast reorientational movement in Mougeotia was inhibited by the calmodulin antagonist trifluoperazine, a hydrophobic compound, or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a hydrophilic compound; 50% inhibition (IC₅₀) of chloroplast movement is caused by 20–50 mol l^-1 trifluoperazine or 100 mol l^-1 W-7. The Ca²⁺-calmodulin may act as an intermediate in the chloroplast reorientational response in Mougeotia governed by phytochrome.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - SDS sodium dodecyl sulfate - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide     

Calmodulin regulates the Ca2+-dependent slow-vacuolar ion channel in the tonoplast of Chenopodium rubrum suspension cells

The patch-clamp technique was applied to vacuoles isolated from a photoautotrophic suspension cell culture of Chenopodium rubrum L. and vacuolar clamp currents, which are predominantly carried by the previously identified Ca²⁺-dependent slow vacuolar (SV) ion channels, were recorded. These currents, which were activated by 1-s voltage pulses of -100 mV (vacuolar interior negative) in the presence of 100 M Ca²⁺ (cytosolic side), could be blocked completely and reversibly by the calmodulin antagonist W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and its chlorine-deficient analogue W-5; half-maximum inhibition was found at approx. 6 M for W-7 and 70 M for W-5. Inhibition was reversed by addition of 1 g · ml^–1 calmodulin purified from Chenopodium cell suspensions; reversal by bovine brain calmodulin was scarcely appreciable. We conclude that cytosolic calmodulin mediates the Ca²⁺ dependence of the SV-channel in the Chenopodium tonoplast.Abbreviations SV-channel slowly activated, vacuolar ion channel - W-5 N-(6-aminohexyl)-1-naphthalenesulfonamide - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide We acknowledge support by the Deutsche Forschungsgemeinschaft and the Bundesminister für Forschung und Technologie, Bonn, and by the Justus-Liebig-Universität Giessen (to W.B.)     

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     

Microfibrillar Structure, Cortical Microtubule Arrangement and the Effect of Amiprophos-methyl on Microfibril Orientation in the Thallus Cells of the Filamentous Green Alga, Chamaedoris orientalis

Microfibrillar structure, cortical microtubule orientation andthe effect of amiprophos-methyl (APM) on the arrangement ofthe most recently deposited cellulose microfibrils were investigatedin the marine filamentous green alga, Chamaedoris orientalis.The thallus cells of Chamaedoris showed typical tip growth.The orientation of microfibrils in the thick cell wall showedorderly change in longitudinal, transverse and oblique directionsin a polar dependent manner. Microtubules run parallel to thelongitudinally arranged microfibrils in the innermost layerof the wall but they are never parallel to either transverseor obliquely arranged microfibrils. The ordered change in microfibrilorientation is altered by the disruption of the microtubuleswith APM. The walls, deposited in the absence of the microtubules,showed typical helicoidal pattern. However, the original crossedpolylamellate pattern was restored by the removal of APM. Thissuggests that cortical microtubules in this alga do not controlthe direction of microfibril orientation but control the orderedchange of microfibril orientation. Amiprophos-methyl, Chamaedoris orientalis, coenocytic green alga, cortical microtubule, microfibrillar structure, tip growth     

Calmodulin and wound healing in the coenocytic green alga Ernodesmis verticillata (Kützing) Børgesen: Ultrastructure of the cortical cytoskeleton and immunogold labeling

Ultrastructural changes in the cortical cytoskeleton during wound-induced cytoplasmic contraction were examined in the coenocytic green alga Ernodesmis verticillata. Both calmodulin (CaM) and actin were localized in intact and contracting cells by immunogold labeling. Within 5 min after wounding, compact microfilament (MF) bundles were observed which increase in diameter as cytoplasmic contraction proceeds. Calmodulin labeling is associated with amorphous material studding the MF bundles, whereas actin labeling occurs along the individual MFs. No MF bundles were ever observed during contraction that were not also labeled with anti-CaM antibodies. In cells treated with the CaM antagonist W-7 (N-[6-aminohexyl]-5-chloro-1-naphtha-lenesulfonamide), MF bundles do not form, and the formation of loosely arranged MFs (similar to nascent bundles in untreated cells) is greatly retarded. We propose that CaM binds indirectly to actin by activating an actin-binding regulatory protein which functions in early stages of the transduction sequence leading to functional MF bundles. Additionally, ultrastructural evidence is presented for a plasma-membrane skeleton or undercoating in this alga.Abbreviations CaM calmodulin - MF(s) microfilament(s) - MT(s) microtubule(s) - W-7 N-[6-aminohexyl]-5-chloro-1-naphthalenesulfonamide We are especially grateful to Dr. J. A. West (University of California, Berkeley, USA) for the original algal isolates and to Dr. L. Van Eldik (Vanderbilt University School of Medicine, Vanderbilt, Tenn., USA) for the generous gift of CaM antibodies. Portions of this work were supported by National Science Foundation grant DCB 84-02345 and U. S. Department of Agriculture grant 87-CRCR-1-2545 to J.W.L.     

Orientation of microfibrils and microtubules in developing tension-wood fibres of Japanese ash (Fraxinus mandshurica var. japonica)

The orientation of cellulose microfibrils (MFs) and the arrangement of cortical microtubules (MTs) in the developing tension-wood fibres of Japanese ash (Fraxinus mandshurica Rupr. var. japonica Maxim.) trees were investigated by electron and immunofluorescence microscopy. The MFs were deposited at an angle of about 45° to the longitudinal axis of the fibre in an S-helical orientation at the initiation of secondary wall thickening. The MFs changed their orientation progressively, with clockwise rotation (viewed from the lumen side), from the S-helix until they were oriented approximately parallel to the fibre axis. This configuration can be considered as a semihelicoidal pattern. With arresting of rotation, a thick gelatinous (G-) layer was developed as a result of the repeated deposition of parallel MFs with a consistent texture. Two types of gelatinous fibre were identified on the basis of the orientation of MFs at the later stage of G-layer deposition. Microfibrils of type 1 were oriented parallel to the fibre axis; MFs of type 2 were laid down with counterclockwise rotation. The counterclockwise rotation of MFs was associated with a variation in the angle of MFs with respect to the fibre axis that ranged from 5° to 25° with a Z-helical orientation among the fibres. The MFs showed a high degree of parallelism at all stages of deposition during G-layer formation. No MFs with an S-helical orientation were observed in the G-layer. Based on these results, a model for the orientation and deposition of MFs in the secondary wall of tension-wood fibres with an S₁ + G type of wall organization is proposed. The MT arrays changed progressively, with clockwise rotation (viewed from the lumen side), from an angle of about 35–40° in a Z-helical orientation to an angle of approximately 0° (parallel) to the fibre axis during G-layer formation. The parallelism between MTs and MFs was evident. The density of MTs in the developing tension-wood fibres during formation of the G-layer was about 17–18 per m of wall. It appears that MTs with a high density play a significant role in regulating the orientation of nascent MFs in the secondary walls of wood fibres. It also appears that the high degree of parallelism among MFs is closely related to the parallelism of MTs that are present at a high density.Abbreviations FE-SEM field emission scanning electron microscopy - G gelatinous layer - MF cellulose microfibril - MT cortical microtubule - S₁ outermost layer of the secondary wall - TEM transmission electron microscopy We thank Dr. Y. Akibayashi, Mr. Y. Sano and Mr. T. Itoh of the Faculty of Agriculture, Hokkaido University, for their experimental or technical assistance.     

Calmodulin and wound healing in the coenocytic green alga Ernodesmis verticillata (Kützing) Børgesen

The involvement of calmodulin (CaM) in wound-induced cytoplasmic contractions in E. verticillata was investigated. Indirect immunofluorescence of CaM in intact cells showed a faint, reticulate pattern of fluorescence in the cortical cytoplasm. Diffuse fluorescence was evident deeper within the cytoplasm. In contracted cells, CaM co-localizes with actin in the cortical cytoplasm in extensive, longitudinal bundles of microfilaments (MFs), and in an actin-containing reticulum. No association of CaM with tubulin was ever observed in the cortical cytoplasm at any stage of wound-healing. When contraction rates in wounded cells are measured, a lag period of 2 min is followed by a rapid, steady rate of movement over the subsequent 10 min. The delay in the initiation of longitudinal contraction corresponds to the time necessary for the assembly of the longitudinal MF bundles. Cytoplasmic motility was inhibited in a dose-dependent manner by CaM antagonists. In these inhibited cells, MF bundles did not assemble, or were poorly formed. In the latter case, CaM was always found associated with MFs. These results indicate a direct spatial and temporal correlation between CaM and actin, and a potential role for CaM in regulating the formation of functional MF bundles during wound-induced cytoplasmic contraction in Ernodesmis.Abbreviations CaM calmodulin - DMSO dimethyl sulfoxide - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - MF(s) microfilament(s) - MT(s) microtubule(s) - TFP trifluoperazine - w-5 N-(6-aminohexyl)-1-naphthalenesulfonamide - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide We are especially grateful to: Dr. J.A. West (University of California, Berkeley) for the original algal isolates; Dr. L. Van Eldik (Vanderbilt University School of Medicine) and Dr. J.L. Lessard (University of Cincinnati College of Medicine) for graciously providing CaM and actin antibodies, respectively; Dr. S.J. Roux (University of Texas, Austin) for the gift of purified oat CaM; Dr.H. Green (Smith, Kline and French Laboratories, Philadelphia, Penn., USA) for providing the trifluoperazine; and M.E.T. Scioli for assistance with the statistical analyses. Portions of this work were supported by National Science Foundation grant DCB 8402345 and U.S. Department of Agriculture grant 87-CRCR-1-2545 to J.W.L.     

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     

Evidence for the involvement of plasma-membrane-bound nitrate reductase in signal transduction during blue-light stimulation of nitrate uptake in Chlorella saccharophila

Nitrate uptake in Chlorella saccharophila (Krüger) Nadson was found to be stimulated by blue light, leading to a doubling of the rate. In the presence of background red light (300 mol photons · m^-2 · s^-1), only 15–20 mol photons · m^-2 · s^-1 of blue light was sufficient to saturate this increased uptake rate. Incubation of Chlorella cells with anti-nitrate-reductase immunoglobulin-G fragments inhibited blue-light stimulation. However, ferricyanide (10 M) doubled and dithiothreitol (100 M) inhibited the stimulatory effect of blue light. Among the protein-kinase inhibitors used, only staurosporine (10 M) prevented the blue-light stimulation. Phosphatase inhibitors were without effect and sodium vanadate totally inhibited nitrate uptake, pointing to an involvement of the plasma-membrane ATPase. Preincubation of the cells with calmodulin antagonists or calcium ionophores did not significantly reduce blue-light stimulation of nitrate uptake. The data are discussed with regard to transduction of the signal for blue-light stimulation of nitrate uptake and the possibility that the plasma-membrane-bound nitrate reductase is the blue-light receptor.Abbreviations Chl chlorophyll - DMSO dimethylsulfoxide - 1,2-DHG 1,2-dihexanoylglycerol - ML-9 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine - NR nitrate reductase - H-7 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine - IgG immunoglobulin G - PFD photon flux density - PM plasma membrane - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide This work was supported by a grant from the Deutsche Forschungs-gemeinschaft to R.T.     

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     

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     

Quantitative radioautographic study of intracellular localization of calmodulin antagonist,W-7, in Chinese-hamster ovary cells

Summary The purpose of the present study was to analyse quantitatively the localization of calmodulin antagonist, n-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) in CHO-Kl cells. The cultured CHO-Kl cells were labelled with 1 (16.7 M), 2 (33.4 M), 5 (83.5 M) and 10 Ci/ml (167 M) tritiated W-7. Some cells were preincubated in 10, 50 and 100 M unlabelled W-7 for 30 min and then labelled with 2 or 5 Ci/ml tritiated W-7 for 1 h. The cells were doubly fixed in glutaraldehyde and osmium-tetroxide solution, and embedded in Epon. For light-microscopic radioautography, 2 m-thick sections were wet mounted with radioautographic emulsion and exposed for 1 month. The radioautograms showed that large numbers of silver grains were mainly localized in the cytoplasm as well as in the nucleus. Quantitative analysis demonstrated that, in both the cytoplasm and nucleus, the number of silver grains was dependent on the concentration of the administered tritiated W-7 and the number was dramatically decreased by the pretreatment of unlabelled W-7. These results show that, in CHO-Kl cells, the W-7 binding sites are saturable. It is concluded that W-7 may get into CHO-Kl cells and be bound to a specific protein that may be calmodulin protein.     

A high-yield procedure for isolation of metaphase chromosomes from root tips ofVicia faba L.

A new method is described for the isolation of large quantities of Vicia faba metaphase chromosomes. Roots were treated with 2.5 mM hydroxyurea for 18 h to accumulate meristem tip cells at the G₁/S interface. After release from the block, the cells re-entered the cell cycle with a high degree of synchrony. A treatment with 2.5 M amiprophos-methyl (APM) was used to accumulate mitotic cells in metaphase. The highest metaphase index (53.9%) was achieved when, 6 h after the release from the hydroxyurea block, the roots were exposed to APM for 4 h. The chromosomes were released from formaldehyde-fixed root tips by chopping with a scalpel in LB01 lysis buffer. Both the quality and the quantity of isolated chromosomes, examined microscopically and by flow cytometry, depended on the extent of the fixation. The best results were achieved after fixation with 6% formaldehyde for 30 min. Under these conditions, 1 · 10⁶ chromosomes were routinely obtained from 30 root tips. The chromosomes were morphologically intact and suitable both for high-resolution chromosome studies and for flow-cytometric analysis and sorting. After the addition of hexylene glycol, the chromosome suspensions could be stored at 4° C for six months without any signs of deterioration.Abbreviations APM amiprophos-methyl - DAPI 4,6-diamidino-2-phenylindole The authors thank Mrs. Jiina Eliáová for her excellent technical assistance and Dr. Slavomir Ondro for the supply of V. faba seeds. A gift sample of APM from the Mobay Corporation (Agricultural Chemicals Division, Kansas City, Mo., USA) is gratefully acknowledged.     

Novel changes in the plasma membrane and cortical cytoplasm during wound-induced contraction in a giant-celled green alga

Summary Changes in the plasma membrane surface and in the cortical cytoplasm during wound healing in giant green algal cells ofErnodesmis verticillata (Kützing) Brgesen were followed using scanning and transmission electron microscopy. Microvillus-like structures that contain cytoplasmic and cytoskeletal constituents were observed emanating from the surface of the plasma membrane at the retracting/cut end of wounded cells. These delicate structures seem to be remnants of cell wall-plasmalemma connections that draw out the plasma membrane and cortical components from the contracting cytoplasm as it pulls away from the cell wall. Most of these connections break during wound healing and, when contraction stops, the microvillus-like protrusions become progressively shorter. In cells treated with a calmodulin antagonist (W-7), a number of distinctive bodies accumulate that are of unknown composition, are oblong in shape, and have a diameter slightly smaller than the protoplasmic protrusions. Ultrastructural and other data indicate that these bodies result from retrieved constituents of the plasma-membrane protrusions, as they do not accumulate in unwounded drugtreated cells or in cells treated in W-5. These findings suggest that the protoplasmic protrusions accumulate membrane and cytoplasmic components that are retrieved and recycled during wound healing inErnodesmis by a novel mechanism. The combined plasma membrane surfaces of the microvillus-like protrusions may help to account for the drastic decrease in surface area that occurs during wound healing.Abbreviations SEM scanning electron microscopy - TEM transmission electron microscopy - W-7 N-[6-aminohexyl]-5-chloro-1-naph-thalenesulfonamide - W-5 N-[6-aminohexyl]-1-naphthalenesulfonamide     

Regulation of the orientation of cortical microtubules inSpirogyra cells

The orientation of cortical microtubules (MTs) was synchronously regulated inSpirogyra cells. While the reorganized MTs in distilled water for 1.5 hr, after 1 hr treatment with amiprophos-methyl (APM) and complete depolymerization of the MTs, were all transverse, those reorganized in 0.30 M mannitol were all oblique or longitudinal. After the MTs had reorganized in 0.30 M mannitol, these cells were then incubated in distilled water for 6 hr, and the orientation of the MTs, in the cells in which MTs could be observed, all became transverse.     

MTOCs in higher plant cells: an immunofluorescent study of microtubule assembly sites following depolymerization by APM

Summary A one hour exposure to 3 M amiprophos-methyl (APM) depolymerizes all MT arrays in cells from higher plant suspension cultures. On removal of APM, MT repolymerization sites are detected using immunofluorescent staining. During interphase, Mt arrays return uniformly dispersed across the cell cortex with transverse arrays in elongated cells and random arrays in isodiametric cells. During cell division, MT arrays return as follows: Prophase-MT arrays return in association with the nuclear envelope. Metaphase-MTs return associated with chromosomes. Teleophase-MTs return in apparent association with the reforming nuclear envelope and as aberrant phragmoplasts. MTOCs in higher plant cells may be membrane associated at many stages in the cell cycle. Isolated, condensed chromosomes are capable of nucleating MTs, which can attain small, spindle-like configurations.Abbreviations APM Amiprophos-methyl - MT Microtubule - MTOC Microtubule organizing center - NS Nucleating site     

Reorganization of the cortical cytoskeleton in tip-growing fern protonemal cells during phytochrome-mediated phototropism and blue light-induced apical swelling

Summary Circular arrays of cortical microtubules (MTs) and microfilaments (MFs) are found in the subapical region of tip-growing protonemal cells of the fernAdiantum capillus-veneris. Reorganization of the two cytoskeletal structures during phytochrome-mediated phototropism and blue light-induced apical swelling was investigated by double-staining of MTs and MFs with rhodaminephalloidin and an indirect immunofluorescence method with tubulinspecific antibody. Before any growth responses were detectable, the MF and MT structures were reorganized according to similar patterns in both photoresponses, that is, oblique orientation and transient disappearance of the structures occurred during the phototropic response, and the disappearance of the structures occurred during apical swelling. The reorganization of MF structures clearly preceded that of the MT structures in the phototropic response. In the case of apical swelling, both types of circular array disappeared with an almost identical time course.These results provide evidence for the significant role of the circular organization of MFs as well as of MTs, in the light-induced growth responses of tip-growing fern protonemal cells. Possible roles of the circular array of MFs in the regulation of tip growth are discussed.Abbreviations DMSO dimethylsulfoxide - PIPES piperazine-N,N-bis(2-ethane-sulfonic acid) - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetraacetic acid - PMSF phenylmethylsulfonyl fluoride - MF microfilament - MT microtubule - Rh-Phal rhodaminelabeled phalloidin     

Inhibitors of cell division and protoplasmic streaming fail to cause a detectable effect on intracellular calcium levels in stamen-hair cells of Tradescantia virginiana L.

The herbicides amiprophos-methyl (APM) and oryzalin disrupt mitosis and cytokinesis in plant cells by causing the depolymerization of microtubules. These drugs have also been shown to affect calcium sequestration by mitochondria. Controversy thus exists as to whether microtubule depolymerization occurs as a result of direct interaction between the drug and tubulin, or because of elevated intracellular calcium levels resulting from drug interference with calcium regulation. In order to clarify this issue we have directly measured the effect of these herbicides and other cell-motility-altering drugs on intracellular calcium levels in stamen-hair cells of Tradescantia. The results indicate that low levels (1–3 M) of APM and oryzalin can act within 3–7 min causing disorganization of mitosis. Studies using the calcium indicator indo-1 injected into stamen-hair cells to monitor internal levels of calcium, show that at drug concentrations where inhibitory effects on mitosis and-or cytokinesis are clearly seen, APM, oryzalin, isopropyl-N-phenyl carbamate, caffeine and cytochalasin D produce no change in intracellular calcium levels. Furthermore, except for cytochalasin D, these drugs do not inhibit cytoplasmic streaming, a calcium-sensitive process. We conclude that the mode of action of these drugs on the cytoskeleton is independent of an effect on intracellular calcium.Abbreviations and Symbols APM amiprophos-methyl - [Ca²⁺]_i free intracellular calcium ion concentration - CD cytochalasin D - DMSO dimethylsulfoxide - IPC isopropyl N-phenylcarbamate - MT(s) microtubule(s) To whom correspondence should be addressedWe thank Dr. L.C. Morejohn, University of Texas, Austin, for encouraging us to perform this study and for his gift of amiprophosmethyl and oryzalin. We also thank our colleagues at the University of Massachusetts for many helpful discussions. This work has been supported by grants from the U.S. Department of Agriculture (88-37261-3727) and the National Science Foundation (DCB-88-01-01750).     

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