Patterns of microtubule reappearance in root cells ofVigna sinensis recovering from a colchicine treatment

Summary The reticulum of paracrystalline tubulin strands, which is assembled in meristematic root cells ofVigna sinensis treated with a 0.08% colchicine solution, disaggregates and microtubules (Mts) reappear after a 10–14 h recovery of the seedlings from the drug. In recovering interphase cells, Mts reappear in the cortical cytoplasm. Initially, they are short and aligned in different directions but finally they elongate and usually become oriented transversely to the long cell axis.A single or a pair of preprophase Mt bands (PMBs) is organized in cells enclosing one or more nuclei. Simultaneously, Mts traverse the perinuclear cytoplasm. In recovering C-mitotic cells, Mt bundles emerge from the kinetochores. Initially, they exhibit diverse orientations. Afterwards, the C-chromosomes are aligned on ametaphase plate via kinetochore Mt bundles, which become parallel to one another. As time passes non-kinetochore Mts appear among the chromosomes and anaphase proceeds. In recovering cytokinetic cells, normal, abnormally curved or branched phragmoplasts are organized. The latter arise between the nuclei of multinucleate telophase cells or between the lobes of forming polyploid nuclei. In cells which were blocked at an advanced cytokinetic stage by colchicine, phragmoplasts return to the margins of the incomplete cell walls.The observations presented here suggest that in recovering colchicine-treated root cells the Mts and the tubulin reticulum are interchangeable. Although Mts appear in cytoplasmic sites where they are expected to be nucleated, the pattern of Mt reformation differs from that operating in normal and to a smaller extent from that functioning in cells recovering from other anti-Mt drugs.     

Colchicine-induced Paracrystals in Root Cells of Wheat (Triticum aestivum L.)

Tubulin conformations other than microtubules in the meristematiccells of wheat roots grown in the presence of 2 mM colchicinesolution were investigated by immunofluorescence and electronmicroscopy. In the affected cells microtubules disappeared andwere replaced by tubulin fluorescent strands that occurred inthe cortical cytoplasm. With increasing time of exposure tocolchicine the tubulin strands became better organized and occurredalso in the subcortical cytoplasm and finally they were restrictedto the area around the nucleus. In prophase and preprophasecells thick strands occupied the cortical cytoplasmic zone wherein normal cells a preprophase microtubule band (PMB) was expectedto be assembled. In the colchicine-treated cells electron microscopy revealedan accumulation of paracrystalline aggregates, which initiallyoccurred along the cell wall and later deeper in the cytoplasm,in the perinuclear regions and the cytoplasmic invaginationsof the nucleus. In transverse planes the paracrystalline strandsappear to consist of hexagonal subunits in a 'honeycomb' arrangement,while in longitudinal and oblique sections they exhibit variableimages. Since their distribution coincides with that of thetubulin strands visualized by immunofluorescence, they are consideredto be the same structure. Therefore, the paracrystals consistof, or at least contain, tubulin. They are most likely to bepolymers of tubulin-colchicine complexes.Copyright 1995, 1999Academic Press Wheat roots, colchicine, immunofluorescence, electron microscopy, tubulin paracrystals, Triticum aestivum L     

Time and cell cycle dependent formation of heterogeneous tubulin arrays induced by colchicine in Triticum aestivum root meristem

We have investigated the appearance and reorganization of tubulin-containing arrays induced by colchicine in the root meristem of wheat Triticum aestivum, using immunostaining and electron microscopy. Colchicine caused depolymerization of microtubules and formation of tubulin cortical strands composed of filamentous material only in C-mitotic cells. After prolonged exposure to the drug, both interphase and C-mitotic cells acquired needle-type bundles, arranged as different crystalloids and/or macrotubules. The unmodified tyrosinated form of alpha-tubulin was detected within microtubules in control cells, but was not found within cortical strands. It was identified, however, within needle-type bundles. The modified acetylated form of alpha-tubulin, which was absent in control cells, was detected within needle-type bundles. Thus, cortical strands were transitory arrays, transformed into needle-type bundles during prolonged exposure to colchicine. Cortical strands appeared in a cell cycle-dependent manner, whereas needle-type bundles were cell cycle stable arrays. The diverse morphological organization, intracellular distribution and stability of tubulin-containing arrays may be associated with heterogeneity of alpha-tubulin isoforms. We assume that non-microtubular arrays substitute for microtubules in conditions where normal tubulin polymerization is inhibited.     

秋水仙素诱导的野生大豆根类细胞超微结构变化

萌发了4d的野生大豆种子经秋水仙素处理3d以后,根尖分生区细胞的超微结构发生了一些显著变化：许多质体环绕细胞核分布,这些质体中含有数量不等的淀粉粒;液泡的体积明显增大,有几个大液泡分布在细胞核周围;少数细胞中形成网状粗面内质网聚集体;细胞核所占细胞体积的比例减小,有的细胞核形状变得极不规则。从超微结构分析,这些细胞已具备了分化细胞的特征,而不再处于有丝分裂之中。     

The Influence of Colchicine on Initiation and Early Development of Adventitious Roots

The first sign of adventitious root formation in the petiole of the primary leaves of Phaseolus vulgaris after treatment with IAA was the dedifferentiation of mature parenchyma cells next to strands of sieve elements and companion cells. Colchicine strongly inhibited this dedifferentiation. Treatment with colchicine 3 days after treatment with IAA, caused the groups of meristematic cells formed to grow by cell enlargement only. Groups of more than about 30 meristematic cells changed into recognizable root primordia during this growth. Groups with a smaller number of meristematic cells extended also in size but did not form a recognizable root primordium.     

Patterns of cortical and perinuclear microtubule organization in meristematic root cells ofAdiantum capillus veneris

Summary The interphase meristematic root cells ofAdiantum capillus venerispossess a well developed cytoskeleton of cortical microtubules (Mts), which disappear at prophase. The preprophase-prophase cells display a well organized preprophase microtubule band (PMB) and a perinuclear Mt system. The observations favour the suggestion that the cell edges included in the PMB cortical zone possess a Mt organizing capacity and thus play an important role in PMB formation. The perinuclear Mts are probably organized on the nuclear surface. The preprophase-prophase nuclei often form protrusions towards the PMB cortical zone and the spindle poles, assuming a conical or rhomboid shape. Mts may be involved in this nuclear shaping.Reinstallation of cortical Mts in dividing cells begins about the middle of cytokinesis with the reappearance of short Mts on the cell surface. When cytokinesis terminates, numerous Mts line the postcytokinetic daughter wall. Many of them converge or form clusters in the cytoplasm occupying the junctions of the new and the old walls. In the examined fern, the cortical Mt arrays seem to be initiated in the cortex of post-cytokinetic root cells. A transitory radial perinuclear Mt array, comparable to that found in post-telophase root cells of flowering plants, was not observed inA. capillus veneris.     

A function of the preprophase band of microtubules in Phleum pratense

Summary A study has been made of the microtubules of the preprophase band and the mitotic spindle in the meristematic cells of the root of Phleum pratense. The preprophase band in these cells is placed symmetrically round the nucleus although a great many of the cells divide asymmetrically. It is suggested that the function of the preprophase band is to orient the nucleus prior to mitosis. The function and formation of the tubules which are found in close association with profiles of smooth endoplasmic reticulum is discussed.     

Cytoskeletal organization and cell organelle transport in basal epithelial cells of the freshwater sponge Spongilla lacustris

Summary Different antibodies against actin, tubulin and cytokeratin were utilized to demonstrate the spatial organization of the cytoskeleton in basal epithelial cells of the freshwater sponge Spongilla lacustris. Accordingly, actin is localized in a cortical layer beneath the plasma membrane and in distinct fibers within the cytoplasmic matrix. Microtubules exhibit a different distributional pattern by radiating from a perinuclear sheath and terminating at, the cell periphery; in contrast, intermediate filaments are lacking. Cytoplasmic streaming activity was studied by in-vivo staining of mitochondria and endoplasmic reticulum by means of fluorescent dyes. Single-frame analysis of such specimens revealed a regular shuttle movement of mitochondria and other small particles between the cell nucleus and the plasma membrane, which can be stopped in a reversible manner with the use of colcemid or colchicine but not with cytochalasin D. The results point to the microtubular system as a candidate for cell organelle transport, whereas the actomyosin system rather serves for changes in cellular shape and motility.     

Cytoskeletal aspects of nuclear migration during tip-growth in the fernAdiantum protonemal cell

Summary In the tip-growing protonemal cell, the nucleus migrates with the tip as it grows, keeping a constant distance between them. Cytoskeletal control of this nuclear migration was analyzed inAdiantum capillus-veneris. Using rhodamine-phalloidin (Rh-Phal), tubulin antibodies and confocal laser scanning microscopy, we found the presence of microtubule (MT) and microfilament (MF) strands connecting the cell nucleus to the cortex of the growing apex. The strands come from the apical end of the spindle-shaped nucleus and run through the endoplasm, arriving at the apical cortex, where a circular arrangement of MTs and MFs is present. Strands of MFs and MTs were also found to emanate from the proximal end of the nucleus and extend towards the cortex of the basal part of the cell. Double staining of MTs and MFs revealed a co-localization of these cytoskeletal elements. When MF strands were disrupted by cytochalasin B (CB), tip-growth ceased and nuclear movement stopped. After the application of colchicine, MT structures disappeared, tip-growth was largely inhibited, and the nucleus moved towards the basal part of the cell. When both CB and colchicine were applied to the cell, no basipetal migration of cell nucleus was observed. These results suggest that the MT strands between the apex and the nucleus may have a role in the anchorage of the cell nucleus to the tip during tip-growth, and that the MF strands may be important for basipetal movement of the nucleus. When the nucleus was dislocated basipetally by centrifugation, cytoskeletal strands between the cell apex and the nucleus were still observed, and by acropetal movement the nucleus resumed its previous position. The acropetal movement of the nucleus was inhibited by the application of both CB and colchicine but not by CB alone nor by colchicine alone, indicating that both cytoskeletal elements are involved in the forward movement of cell nucleus.Abbreviations CB cytochalasin B - DAPI4 6-diamino-2-phenylin-dole - DMSO dimethylsulfoxide - PIPES piperazine-N,N-bis(2-ethane-sulfonic acid) - EGTA ethyleneglycol-bis-(-aminoethyl-ether)-N,N,N,N-tetraacetic acid - MBS m-maleimidobenzoic acid N-hydroxysuccinimide ester - MF microfilament - MT microtubule - PMSF phenylmethylsulfonyl fluoride - PSM polyoxyethylene sorbitan monolaurate - Rh-Phal rhodamine-labeled phalloidin     

淹水玉米幼苗根尖分生细胞内Ca2+超微细胞化学定位

采用焦锑酸钾沉淀法,对遭受淹水胁迫的玉米幼苗初生根根尖分生细胞内钙离子分布变化情况进行了电镜细胞化学观察。在正常状态下,根尖分生细胞内Ca^2＋沉淀颗粒的分布较少．主要位于细胞核和细胞质中。在淹水1h后,根尖分生细胞内呈现有大量Ca^2＋沉淀颗粒分布,细胞核和细胞质中分布的Ca^2＋沉淀颗粒密度,远大于正常细胞。随着淹水时间的延长,根尖分生细胞的细胞核和细胞质中分布的Ca^2＋沉淀颗粒呈现不断增多的趋势,而液泡中分布的Ca^2＋沉淀颗粒则逐步明显减少。根据实验结果本文对受淹根尖分生细胞的死亡与Ca^2＋分布变化的关系进行了研究。     

Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells

Cells were prepared for indirect immunofluorescence microscopy after paraformaldehyde fixation of multicellular root apices and brief incubation in cell wall-digesting enzymes. This allowed subsequent separation of the tissue into individual cells or short files of cells which were put onto coverslips coated with polylysine. Unlike spherical protoplasts made from living tissues, these preparations retain the same polyhedral shape as the cells from which they are derived. Cellular contents, including organized arrays of microtubules, are likewise structurally stabilized. Antibodies to porcine brain tubulin react with all types of microtubule array known to occur in plant meristematic cells, namely, interphase cortical microtubules, pre- prophase bands, the mitotic spindle, and phragmoplast microtubules. The retention of antigenicity in permeabilized, isolated, stabilized cells from typical, wall-enclosed plant cells has much potential for plant immunocytochemistry, and in particular should facilitate work on the role of microtubules in the morphogenesis of organized plant tissues.     

Colchicine-resistant assembly of tubulin in plant mitosis

Summary Tubulin distribution in c-mitoses (induced by 1 mM colchicine) has been studied by indirect immunofluorescence with monoclonal antibodies inAllium cepa L. meristems proliferating under steady state kinetics. Two hours after colchicine treatment was initiated tubulin is detected in approximately 25% of the cells as arrowheads on the kinetochores, as if these structures stabilize microtubules against disassembly. Total disassembly of microtubules occurs in 70% of the c-mitoses six hours after the initiation of the colchicine treatment, when restitution nuclei also start appearing. After 2 to 14 hours of colchicine treatment, tubulin is detected in about 30% of the c-mitoses, both in small kinetochores-like dots and in a strand which apparently connects sister kinetochores. Other larger microtubule-like structures, up to 20 m long, apparently unassociated with kinetochores, are assembled in the presence of cholchicine in c-mitoses after 10 hours. Such structures disappear when chromosomes decondense and the nuclear envelope reforms in the restitution nucleus; they do not seem to be related to interphase cortical microtubules which reappear in control telophase.     

Organization of microtubules in stabilized meristematic plant cells revealed by a rat monoclonal antibody reacting only with the tyrosinated form of alpha-tubulin

A rat monoclonal antibody against yeast tubulin (clone YL 1/2; Kilmartin et al., 1982) that reacts specifically with mammalian alpha-tubulin carrying a carboxyterminal tyrosine residue (Wehland et al., 1983) was used to localize microtubules in plant cells derived from onion root apices (Allium cepa L.). YL 1/2 reacted with all types of microtubular arrays known to occur in higher plant meristematic cells such as interphase cortical microtubules, pre-prophase bands, the mitotic spindle and phragmoplast microtubules. The specific labeling of microtubules in isolated cells from onion root tips by YL 1/2 indicates that plant cells like animal cells contain tubulin tyrosine ligase, the enzyme which posttranslationally modifies alpha-tubulin. This enzyme could be involved in the dynamic regulation of microtubular arrays in all eukaryotic cells.     

Differential Responsiveness of Cortical Microtubule Orientation to Suppression of Cell Expansion among the Developmental Zones of Arabidopsis thaliana Root Apex

Τhe bidirectional relationship between cortical microtubule orientation and cell wall structure has been extensively studied in elongating cells. Nevertheless, the possible interplay between microtubules and cell wall elements in meristematic cells still remains elusive. Herein, the impact of cellulose synthesis inhibition and suppressed cell elongation on cortical microtubule orientation was assessed throughout the developmental zones of Arabidopsis thaliana root apex by whole-mount tubulin immunolabeling and confocal microscopy. Apart from the wild-type, thanatos and pom2-4 mutants of Cellulose SynthaseA3 and Cellulose Synthase Interacting1, respectively, were studied. Pharmacological and mechanical approaches inhibiting cell expansion were also applied. Cortical microtubules of untreated wild-type roots were predominantly transverse in the meristematic, transition and elongation root zones. Cellulose-deficient mutants, chemical inhibition of cell expansion, or growth in soil resulted in microtubule reorientation in the elongation zone, wherein cell length was significantly decreased. Combinatorial genetic and chemical suppression of cell expansion extended microtubule reorientation to the transition zone. According to the results, transverse cortical microtubule orientation is established in the meristematic root zone, persisting upon inhibition of cell expansion. Microtubule reorientation in the elongation zone could be attributed to conditional suppression of cell elongation. The differential responsiveness of microtubule orientation to genetic and environmental cues is most likely associated with distinct biophysical traits of the cells among each developmental root zone.     

In vitro stimulation and inhibition of steroid hormone release from postovulatory follicles of the tilapia,Oreochromis mossambicus

Summary Postovulatory follicles of the tilapia, Oreochromis mossambicus, were incubated with graded doses of salmon gonadotropin to identify the steroid hormones released by this tissue. In addition, the effects of either cytochalasin B or colchicine on steroid hormone release were studied. After the incubation, the tissue was examined by electron microscopy. Postovulatory follicles released testosterone and estradiol-17B in a dose-dependent manner with gonadotropin. There was no detectable release of progesterone or 17a-OH-progesterone. When stimulated with high doses of gonadotropin, the steroidogenic cells showed an increase in smooth endoplasmic reticulum, Golgi complexes, and lipid droplets. Also, microfilaments became arranged in orderly bundles and were found close to the numerous secretory vesicles and lipid droplets. Upon incubation with gonadotropin and either colchicine or cytochalasin B, the cells still appeared steroidogenic, but the filaments were not organized nor associated with vesicles or lipid droplets. Release of steroid hormone decreased significantly. Also in these tissues, vesicles were no longer numerous in the apical region of the granulosa cells, but were located primarily near smooth endoplasmic reticulum and Golgi complexes. This suggests that disruption of the cytoskeleton results in reduced steroid hormone synthesis or release.     

Paracrystalline structures in the epithelial principal cells of the epididymis of water buffalo (Bubalus bubalis).

The ultrastructure of many principal cells in the cauda epididymis of water buffaloes with ages varying between 4, 18, 24, 30 and 36 months, revealed, in many cells, the presence of long, curved paracrystalline structures that are quite large and frequently encountered in the cytoplasm, usually near the nucleus. Concomitantly or not, smaller rod-like, hexagonal or curled structures can be found in the nucleus. Both structures, cytoplasmic and intranuclear, are made up of a sheath of parallel filaments. These paracrystals may appear as thin, regularly spaced filaments that are associated with fine, evenly spaced subunits. Occasionally, the association of paracrystalline structures with membranes similar to the endoplasmic reticulum was observed, but no membranes were consistently found in close contact with the nuclear crystalloids. It is postulated that both structures are proteinaceous and may represent stored enzymes or substances present in the intraluminal fluid, which are absorbed and initially stored in numerous intraepithelial vacuoles of the corpus and cauda of the buffalo epididymis.     

Topographical features of the membrane of Poterioochromonas malhamensis after colchicine and osmotic treatment

Changes in membrane topography in the flagellate Poterioochromonas malhamensis, as a result of colchicine and osmotic-stress treatments, have been studied using freeze-fracturing and thin sectioning. Ridges, but not rows of intramembrane particles, in the PF-face which denote the position of underlying cortical microtubules, together with the ridge associated with their point of origin (flagellar root fibre 1), dissappear after colchicine or short-term (5 min) osmotic treatments. Cortical microtubules are destroyed as a result of the former, but not the latter treatment. Longer periods in osmoticum allow a recovery of the microtubule — associated membrane ridges. Despite careful isosmotic fixations distinct cross-bridges between microtubules and the plasmalemma were not discernible in thin section.     

Fertilization-induced changes in the microtubular architecture of the maize egg cell and zygote—an immunocytochemical approach adapted to single cells

By using single cell micromanipulation techniques, we developed an immunocytochemical procedure to examine subcellular protein localization in isolated and cultured cells. Localization of microtubules was examined in isolated single egg cells and developing zygotes of maize with anti--tubulin antibodies. In egg cells, a few cortical microtubules were detected but well organized microtubules were rarely observed. In contrast, distinct cortical microtubules and strands of cytoplasmic microtubules radiating from the nucleus to the cell periphery were observed in developing zygotes. Solely cortical microtubules were observed in zygotes up to 7 h after in vitro fertilization. After this time, radiating microtubules additionally appeared, and persisted during zygote development. These results indicate early and pronounced fertilization-induced changes in microtubular organization in the fertilized egg cell of maize.     

A mycorrhizal fungus changes microtubule orientation in tobacco root cells

Summary Cortical cells of mycorrhizal roots undergo drastic morphological changes, such as vacuole fragmentation, nucleus migration, and deposition of cell wall components at the plant-fungus interface. We hypothesized that the cytoskeleton is involved in these mechanisms leading to cell reorganization. We subjected longitudinal, meristem to basal zone, sections of uninfectedNicotiana tabacum roots to immunofluorescence methods to identify the microtubular (MT) structures associated with root cells. Similar sections were obtained from tobacco roots grown in the presence ofGigaspora margarita, an arbuscular mycorrhizal fungus which penetrates the root via the epidermal cells, but mostly develops in the inner cortical cells. While the usual MT structures were found in uninfected roots (e.g., MTs involved in mitosis in the meristem and cortical hoops in differentiated parenchyma cells), an increase in complexity of MT structures was observed in infected tissues. At least three new systems were identified: (i) MTs running along large intracellular hyphae, (ii) MTs linking hyphae, (iii) MTs binding the hyphae to the host nucleus. The experiments show that mycorrhizal infection causes reorganization of root MTs, suggesting their involvement in the drastic morphological changes shown by the cortical cells.     

Morphogenesis of the protocorm ofCypripedium acaule (Orchidaceae)

The nature of the protocorm of theOrchidaceae has fascinated morphologists for more than a century. In the present study, the development of the protocorm was followed using in vitro germination of seeds on a culture medium containing sugar, but without a symbiont. Inside the seed, the embryo consists of about a hundred cells. In the embryo, cells are arranged along a longitudinal axis according to size; these cells contain protein and lipid reserve material. In the first stages of seedling development, the embryo is transformed into a protocorm and meristematic tissue becomes organized into a meristematic dome (promeristem) at the anterior pole. This meristematic dome will give rise to a scale and the apex of the seedling. At first, the apex and the scale leaf develop synchronously. The development of the root always follows that of the apex. The study of the development of the seed ofCypripedium acaule showed that the protocorm is a distinct morphological system with respect to the rest of the cormus. The protocorm may be interpreted as an extension of the proembryonic stage.