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.     

Assembly of cortical microtubules during cold treatment of the coenocytic green alga,Chaetomorpha moniligera

The effects of cold treatment on the cortical microtubules (MTs) of Chaetomorpha moniligera Kjellman were investigated by immunofluorescence microscopy. Cortical MTs in Chaetomorpha thallus are arranged longitudinally. In this study, 70–75% of MTs disassembled within 4 h on ice while the others remained stable under these conditions. Reticulate background immunofluorescence, assumed to indicate the presence of a tubulin monomer, was distributed about the stable MTs. Immunofluorescence was prominent in only 50% of the cells. Tubulin polymerization was noted where the background and MT immunofluorescence was strong. New MTs grew transversely as single strings or clusters from the sides of MTs after cold treatment for 4 h and elongated with time to take on a reticulate form at 24 h. The significance of this tubulin polymerization under cold treatment is discussed.Abbreviations MT microtubule - MTOC microtubule-organizing center     

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     

Dynamics of microtubule reassembly and reorganization in the coenocytic green alga Ernodesmis verticillata (Kützing) Børgesen

The dynamics of microtubule (MT) disassembly and reassembly were studied in the green alga Ernodesmis verticillata, using indirect immunofluorescent localization of tubulin. This alga possesses two distinct MT arrays: highly-ordered, longitudinally-oriented cortical MTs, and shorter perinuclear MTs radiating from nuclear surfaces. Perinuclear MTs are very labile, completely disassembling in the cold (cells on ice) within 5–10 min or in 25 μM amiprophos-methyl (APM) within 15–30 min. Although cortical MTs are generally absent after 3 h in APM, it takes 45–60 min before any cold-induced depolymerization is apparent, and some cortical MTs persist after 6 h of cold treatment. The extent of immunofluorescence of cytoplasmic (depolymerized?) tubulin is inversely proportional to the abundance of cortical MTs. Recovery of MT arrays upon warming or upon removal of APM occurs within 30–60 min for the perinuclear MTs, but the cortical arrays take much longer to regain their normal patterns. The cortical MTs initially reappear in a random distribution with respect to the cell axis, but within 3–4 d of warming (or 24–36 h of removing APM) they are nearly parallel to each other and to the cell's longitudinal axis. Thus, although the timing differs, the actual patterns of depolymerization and recovery are similar, irrespective of whether physical or chemical agents are used. Longer-term treatments in 1 μM APM indicate that despite the rapid disappearance of perinuclear MTs, a loss of the uniform nuclear spacing occurs gradually over 1–6 d. Similar disorganization of nuclei is obtained with long-term treatment with 1 μM taxol, where a gradual loss of perinuclear MTs is accompanied by an increased abundance of mitotic spindles. This implies that perinuclear MTs can disassemble in vivo in the presence of taxol, and that they are not the sole components involved in maintaining nuclear spacing in these coenocytes. The results indicate that both nuclear and cortical sites of MT nucleation may exist in this organism, and that MT reassembly and re-organization are temporally distinct events in cells that have highly-ordered arrays of long MTs.     

Re-Formation and Ordering of Wall Microtubules in Spirogyra Cells

Processes of re-formation and ordering of wall microtubules(wall MTs) in Spirogyra cells were examined using immunofluorescencemicroscopy. Wall MTs were usually arranged nearly transverselyto the cell axis at all stages of the cell cycle. Two-hour treatmentwith amiprophosmethyl (APM) completely disrupted wall MTs, butremoval of the APM led to re-formation of randomly orientedwall MTs at many sites over the cell. The re-formed wall MTsgradually assumed a transversely oriented order without anyspecific MT-ordering centers, indicating that initiation andordering of MTs are different processes. Removal of APM after24-h treatment caused reformation of randomly oriented wallMTs, followed in some cells by gradual ordering to obliquelyoriented instead of transversely oriented wall MTs. This orderingoccurred with the same sign of obliquity as that of chloroplastspirals. When cells were centrifuged along the cell axis, chloroplastssedimented on the cross wall, but the transverse wall MTs didnot. In centrifuged cells, wall MTs were re-formed and orderedtransversely after MT depolymerization by APM for 2 h as innon-centrifuged cells. When cells were centrifuged for the final30 min in 2-h treatment with APM, wall MTs that re-formed afterremoval of APM were sometimes ordered transversely over thatpart of the cell which contained sedimented chloroplasts, butremained at random over the other part, as though the MT-orderingfactor was sedimented by the centrifugation. The mechanism determiningthe wall MT orientation is discussed. (Received January 12, 1987; Accepted April 27, 1987)     

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     

Reinstatement of Microtubule Arrays from Cortical Nucleating Sites in Stomatal Complexes of Lolium rigidum Following Depolymerization of Microtubules by Oryzalin and High Pressure

Immunofluorescence visualization of microtubule (MT) arraysin stomatal complexes of Lolium rigidum shows that disassemblyof the arrays can be successfully achieved using oryzalin orhigh pressure treatments. Under conditions allowing for MT recovery,MTs reappear within an hour after oryzalin or within 5 min afterhigh pressure treatment. During recovery guard mother cells(GMCs) nucleate MTs at sites distributed randomly in the cellcortex. Even after 22 h of recovery the MTs are not arrangedinto any configuration found in untreated tissue. This inabilityto reorganize their MTs after treatment makes GMCs more sensitiveto the loss of MTs than are other cells of the leaf. In guardcells (GCs) MTs reappear around the pore at the junction ofthe periclinal and ventral walls. They subsequently appear throughoutmost of the cell cortex and the majority of stomatal complexesrecover fully organized MT arrays indistinguishable from thosein untreated cells. The results support and extend ultrastructuraland immunofluorescence observations that suggest that MTs inGCs of developing stomata are nucleated in the cell cortex. ²Present address: Department of Biology, The University of SouthwesternLouisiana, Lafayette, Louisiana 70504-2451, U.S.A. (Received April 24, 1990; Accepted July 7, 1990)     

Cadmium effects on the organization of microtubular cytoskeleton in interphase and mitotic cells of Allium sativum

We have investigated the effects of cadmium on the microtubular (MT) cytoskeleton in the root tip cells of Allium sativum L. using indirect immunofluorescence microscopy. Cd affected the mechanisms controlling the organization of MT cytoskeleton, as well as tubulin assembly/disassembly processes. Cd induced the formation of abnormal MT arrays, consisting of discontinuous wavy MTs or short MT fragments at the cell periphery. Cadmium caused irregular nuclear disorder in cells where the MT organization and function was disturbed. Furthermore, with increased Cd concentration and duration of treatment the MTs depolymerized more severely, the frequency of abnormal cell increased and the mitotic index decreased progressively. The above findings showed that MT cytoskeleton is one of target sites of Cd toxicity in root tip cells.     

Modification in Cell Shape Unrelated to Cellulose Microfibril Orientation in Growing Thallus Cells of Chaetomorpha moniligera

Cellulose microfibril orientation patterns in thallus cellsof Chaetomorpha moniligera were studied, and the relationshipbetween the microfibril and the peripheral microtubule arrangementsduring cell-shape modification by colchicine was examined. Inthe cuttings from growing thalli, linearly arranged cylindricalcells developed into cask-shaped cells during 4–6 daysof culture at 27?C. In the cylindrical cells, microfibrils formingthe innermost portion of the wall were arranged alternatelyin longitudinal and transverse directions, but peripheral microtubuleswere always arranged only in a longitudinal direction. Thesefeatures were also noted in the cask-shaped cells. Colchicineat 10^–3M and 3?10^–3M accelerated both cell expansionand wall thickening with matrix deposition, but the directionsin which both microfibrils and microtubules were arranged werethe same as those of the cylindrical cells. These results indicatethat (1) the microfibril and microtubule arrangements of Chaetomorphaare not necessarily correlated, (2) changes in cell shape ofChaetomorpha are not necessarily accompanied by changes in thearrangement of cell-wall microfibrils, and (3) colchicine playsa role in the loosening and thickening of cell walls by enhancingmatrix deposition. (Received June 2, 1986; Accepted February 13, 1987)     

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     

Complete disintegration of the microtubular cytoskeleton precedes its auxin-mediated reconstruction in postmitotic maize root cells

The inhibitory action of 0.1 microM auxin (IAA) on maize root growth was closely associated with a rapid and complete disintegration of the microtubular (MT) cytoskeleton, as visualized by indirect immunofluorescence of tubulin, throughout the growth region. After 30 min of this treatment, only fluorescent spots were present in root cells, accumulating either around nuclei or along cell walls. Six h later, in addition to some background fluorescence, dense but partially oriented oblique or longitudinal arrays of cortical MTs (CMTs) were found in most growing cells of the root apex. After 24 h of treatment, maize roots had adapted to the auxin, as inferred from the slowly recovering elongation rate and from the reassembly of a dense and well-ordered MT cytoskeleton which showed only slight deviations from that of the control root cells. Taxol pretreatment (100 microM, 24 h) prevented not only the rapid auxin-mediated disintegration of the MT cytoskeleton but also a reorientation of the CMT arrays, from transversal to longitudinal. The only tissue to show MTs in their cells throughout the auxin treatment was the epidermis. Significant resistance of transverse CMT arrays in these cells towards auxin was confirmed using a higher auxin concentration (100 microM, 24 h). The latter auxin dose also revealed inter-tissue-specific responses to auxin: outer cortical cell files reoriented their CMTs from the transversal to longitudinal orientation, whereas inner cortical cell files lost their MTs. This high auxin-mediated response, associated with the swelling of root apices, was abolished with the pretreatment of maize root with taxol.     

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     

Metabolic inhibitors and mitosis: II. Effects of dinitrophenol/deoxyglucose and nocodazole on the microtubule cytoskeleton

Summary To examine the effects exerted on the microtubule (MT) cytoskeleton by dinitrophenol/deoxyglucose (DNP/DOG) and nocodazole, live PtK₁ cells were treated with the drugs and then fixed and examined by immunofluorescence staining and electronmicroscopy. DNP/DOG had little effect on interphase MTs. In mitotic cells, kinetochore and some astral fibers were clearly shortened in metaphase figures by DNP/DOG. Nocodazole rapidly broke down spindle MTs (except those in the midbody), while interphase cells showed considerable variation in the susceptibility of their MTs. Nocodazole had little effect on MTs in energy-depleted (DNP/DOG-treated) cells. When cytoplasmic MTs had all been broken down by prolonged nocodazole treatment and the cells then released from the nocodazole block into DNP/DOG, some MT reassembly occurred in the ATP-depleted state. MTs in permeabilized, extracted cells were also examined with antitubulin staining; the well-preserved interphase and mitotic arrays of MTs showed no susceptibility to nocodazole. In contrast, MTs suffered considerable breakdown by ATP, GTP and ATPS; AMPPNP had little effect. This susceptibility of extracted MT cytoskeleton to nucleotide phosphates was highly variable; some interphase cells lost all MTs, most were severely affected, but some retained extensive MT networks; mitotic spindles were diminished but structurally coherent and more stable than most interphase MT arrays.We suggest that: 1. in the living cell, ATP or nucleotide triphosphates (NTPs) are necessary for normal and nocodazole-induced MT disassembly; 2. the NTP requirement may be for phosphorylation; 3. shortening of kinetochore fibers may be modulated by compression and require ATP; 4. many of these results cannot be accomodated by the dynamic equilibrium theory of MT assembly/disassembly; 5. the use and role of ATP on isolated spindles may have to be reevaluated due to the effects ATP has on the spindle cytoskeleton of permeabilized cells.     

Role of plasma membrane proteins and cell wall in meridional arrangement of cortical microtubules inBoodlea coacta

Summary The effects of 2,6-dichlorobenzonitrile (DCB, an agent which inhibits cellulose synthesis) and cycloheximide (CHI, a known inhibitor of protein synthesis) on the construction and stability of the cortical microtubule (MT) cytoskeleton in two kinds of protoplasts (smaller protoplasts and larger ones) prepared fromBoodlea coacta (Dickie) Murray et De Toni were examined by immunofluorescence microscopy. In smaller protoplasts which develop from released protoplasmic masses in culture media, parental cortical MTs assume a convoluted configuration, but new cortical MTs appear following disassembly of convoluted MTs. New cortical MTs initially have a random arrangement but later, a rough meridional arrangement following development of cell polarity and finally, a high density meridional arrangement. In larger protoplasts which are formed within cell wall cylinders of thalli cut at 500 m length, longitudinally oriented parental cortical MTs are preserved. Each exhibits a curving configuration just after protoplast formation, but a straight configuration after 3 h of culture. In smaller protoplasts, cortical MT orientation changes from random to rough meridional orientation but never to a high density meridional orientation following treatment with 10 M CHI, and MT density decreases after 12 h. However, rough meridional and high density meridional arrangements of MTs ceased to be formed and MT density decreased following treatment with 10 M DCB. In larger protoplasts, high density meridional arrangements of MTs were noted not to be affected by treatment with CHI; instead, they continued to remain oriented meridionally, but the length and density were decreased after treatment with DCB for 3–4 h. After 10 h, the MTs became fragmented and orientation was random. From these findings it is summarized that: (1) There are no putative anchors in the plasma membrane of nascent smaller protoplasts, but the meridional orientation of cortical MTs requires anchors which may be distributed in the plasma membrane following the establishment of cell polarity. (2) Plasma membranes in larger protoplasts contain parental anchors oriented meridionally. Anchors stabilize cortical MTs via their close relation to cell walls (especially to cellulose). Anchors are detached from the plasma membrane when cellulose is not formed. (3) Cellulose regeneration may be indispensable to the formation and stabilization of the MT cytoskeleton inBoodlea.Abbreviations CHI cycloheximide - DCB 2,6-dichlorobenzonitrile - DMSO dimethylsulfoxide - MT microtubule     

The microtubular cytoskeleton during development of the zygote,proembryo and free-nuclear endosperm inArabidopsis thaliana (L.) Heynh

The microtubular cytoskeleton has been studied during development of the zygote, proembryo and free-nuclear endosperm inA. thaliana using immunofluorescence localization of tubulin in enzymatically isolated material. Abundant micro tubules (MTs) are found throughout proembryogenesis. Microtubules in the coenocytic endosperm are mainly internal. By contrast, there is a re-orientation of MTs to a transverse cortical distribution during zygote development, predominantly in a subapical band which accompanies a phase of apical extension. The presence of these cortical arrays coincides with the elongation of the zygote. Cortical arrays also accompany elongation of the cylindrical suspensor. Extensive networks of MTs ramify throughout the cytoplasm of cells in the proembryo proper. Perinuclear arrays are detected in a number of cell types and MTs contribute to typical mitotic configurations during nuclear divisions. Preprophase bands of MTs are absent throughout megasporogenesis and embryo-sac development and do not occur in endosperm cell divisions. We have observed MTs throughout the first division cycle of the zygote. By placing the observed stages in a most probable sequence, we have identified this cell cycle as the point during embryogenesis at which a preprophase band is reinstated as a regular feature of cell division. Preprophase bands were observed to predict planes of cytokinesis in cell divisions up to the octant stage.Abbreviations DIC differential interference contrast optics - MT microtubule - PPB preprophase band of microtubule We thank Ms. Margaret Travers for her helpful English translation of Yakovlev and Alimova (1976) and Mr. James Whitehead for preparation of Fig. 11. M.C.W. was supported by an Australian Postgraduate Research Award.     

LEARNING AS A FACTOR IN DIET SELECTION BY ELYSIA VIRIDIS (MONTAGU) (OPISTHOBRANCHIA).

Elysia viridis (Montagu) occurs naturally in populations feedingon the fleshy, siphonalean green alga Codium or on the coarse,filamentous green alga Chaetomorpha. Only about half of theE. viridis transferred from Codium to Chaetomorpha in the laboratorywere able to learn to feed on this alga. Handling time decreasedsignificantly during the learning-period. All E. viridis transferredfrom Chaetomorpha to Codium in the laboratory were able to learnto feed on the latter alga. Handling time also decreased withexperience in these animals. Ingestive conditioning occurredin animals transferred from Chaetomorpha to Codium. These animalshad to go through a learning-period again when they were offeredChaetomorpha after 6–9 weeks with Codium Ingestive conditioningwas also evident from preference experiments; animals preferredthe food they had been kept on prior to the experiments. Thesefindings are discussed in relation to optimal diet theory. (Received 14 December 1987; accepted 22 March 1988)     

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     

Pb/Cu effects on the organization of microtubule cytoskeleton in interphase and mitotic cells of Allium sativum L.

The effects of lead and copper on the arrangement of microtubule (MT) cytoskeleton in root tip cells of Allium sativum L. were investigated. Batch cultures of garlic were carried out under defined conditions in the presence 10⁻⁴ M Pb/Cu of various duration treatments. With tubulin immunolabelling and transmission electron microscopy (TEM), we found four different types of MT structures depending on the cell cycle stage: the interphase array, preprophase band, mitotic spindle and phragmoplast were typical for the control cells. Pb/Cu affected the mechanisms controlling the organization of MT cytoskeleton, and induces the following aberrations in interphase and mitotic cells. (1) Pb/Cu induced the formation of atypical MT arrays in the cortical cytoplasm of the interphase cells, consisting of skewed, wavy MT bundles, MT fragments and ring-like tubulin aggregations. (2) Pb/Cu disordered the chromosome movements carried out by the mitotic spindle. The outcome was chromosome aberrations, for example, chromosome bridges and chromosome stickiness, as well as inhibition of cells from entering mitosis. (3) Depending on the time of exposure, MTs disintegrated into shorter fragments or they completely disappeared, indicating MT depolymerization. (4) Different metals had different effects on MT organization. MTs were more sensitive to the pressure of Cu ions than Pb. Moreover, TEM observations showed that the MTs were relatively short and in some places wavy when exposed to 10⁻⁴ M Pb/Cu solutions for 1–2 h. In many sections MTs were no longer visible with increasing duration of treatment (>4 h). Based on these results, we suggested that MT cytoskeleton is primarily responsible for Pb/Cu-associated toxicity and tolerance in plants.     

植物微管特效解聚剂APM对紫露草雄蕊毛细胞胞质环流的影响

采用体外渗透和显微注射的方法。将植物微管特效解聚剂甲基氨草磷(APM)引入紫露草雄蕊毛细胞后,发现原来沿着胞质束运动的胞质颗粒运动速度渐慢,进而胞质束消失,颗粒运动停止。显微注射后,还发现APM可通过胞间通道由被注射的细胞向两侧细胞扩散,从而也导致两侧细胞胞质束消失,颗粒运动停止。APM对胞质环流的抑制作用是可逆的。结果表明微管可能是胞质束的重要组份之一,植物胞质环流与微管的聚合与解聚状态有密切关系。     

The cytoskeleton of neurites after microtubule depolymerization

We previously reported a positive correlation between the number of cold-stable microtubules (MTs) remaining after cold treatment of cat sympathetic nerve and the extent to which the original uniform polarity orientation of axonal MTs was recapitulated after rewarming (J cell biol 99 (1984) 1289). We interpreted these data to indicate that cold-stable fragments, part of larger, generally labile MTs, could act as seeds to organize MT assembly in axons. We report here a direct investigation of the form of cold-stable MTs in neurites of PC-12 cells using two-dimensional reconstruction of serial thin sections. Our data provides strong support for our previous interpretation. The number of MTs in cold-treated neurites was 2-3 times as great while the total length of polymer was approximately half that in control neurites. The average length of MTs in cold-treated neurites was 7-10 times lower than in control neurites. We observed that treatments that depolymerize axonal microtubules cause a marked increase in the number of membranous elements within the axoplasm. This may, however, be a non-specific result of an insult to the axon, since such changes have also been observed in severed, regenerating nerve fibres. We observed that neuroblastoma neurites respond to MT-depolymerization stimuli by developing lateral filopodia similar to those observed in chick dorsal root ganglion cells. Ultrastructural observation of detergent-lysed, whole mounted neuroblastoma (Neuro 2A) cells indicated that the cytoskeleton remaining after MT depolymerization splayed out perpendicular to the long axis of the neurite. That is, we were able to observe many more cytoskeletal 'ends' after MT depolymerization. The concomitant production of filopodia and the splaying of the cytoskeleton after MT depolymerization supports the hypothesis put forward by Wessels et al. (Exp cell res 117 (1978) 335) that the presence or absence of cytoskeletal ends regulates which region of the cell surface is involved in motile behaviour.