Effects of methylmercury on retinoic acid-induced neuroectodermal derivatives of embryonal carcinoma cells

Immunofluorescence staining with antibodies to tubulin, neurofilaments and glial filaments was used to study the effects of methylmercury on the differentiation of retinoic acid-induced embryonal carcinoma cells into neurons and astroglia and on the cytoskeleton of these neuroectodermal derivatives. Methylmercury did not prevent undifferentiated embryonal carcinoma cells from developing into neurons and glia. Treatment of committed embryonal carcinoma cells with methylmercury doses exceeding 1 M resulted in the formation of neurons with abnormal morphologies. In differentiated cultures, microtubules were the first cytoskeletal element to be affected. Their disassembly was time- and concentration-dependent. Microtubules in glial cells and in neuronal perikarya were more sensitive than those in neuronal processes. Neurofilaments and glial filaments appeared relatively insensitive to methylmercury treatment but showed reorganization after complete disassembly of the microtubules. The data demonstrate 1) the sensitivity of microtubules of both neurons and glia to methylmercury-induced depolymerization, and 2) the heterogeneous response of neuronalAbbreviations -MEM alpha minimal essential medium - EC embryonal carcinoma cells - FCS fetal calf serum - MAP microtubule-associated protein - MeHg methylmercury - RA retinoic acid     

γ-Tubulin in tobacco pollen tubes: association with generative cell and vegetative microtubules

-Tubulin was localized in tobacco pollen tubes using an antibody raised against a peptide conserved in all known -tubulins. Antibody staining occurs in a primarily punctate pattern along the length of the microtubule bundles in generative cells and along cortical microtubules in the vegetative cytoplasm. During generative cell division, -tubulin is localized in the forming mitotic apparatus. By metaphase, it is present along kinetochore fibers except at their plus ends located at the kinetochores. By telophase, staining is observed in the phragmoplast, where it again avoids the plus ends of microtubules at the cell plate. -Tubulin is also present at the periphery of the sperm nuclei. A patch of intense staining on the distal side of each nucleus marks the site of assembly of a new population of sperm microtubules. No specific fluorescence is present in control pollen tubes treated with preimmune IgG. These localization patterns bear similarities to those seen in somatic cells and in addition may help explain changes in microtubule arrays between generative cells and sperm.     

Relationship between 1-chloro2,4-dinitrobenzene-induced cytoskeletal perturbations and cellular glutathione

Exposure of 3T3 cells to micromolar doses of 1-chloro-2,4-dinitrobenzene, a substrate for glutathione-S-transferase, resulted in a rapid depletion of total cellular glutathione accompanied by disassembly of microtubules as visualized by fluorescence microscopy. However, prolonged incubation resulted in cellular recovery from 1-chloro-2,4-dinitrobenzene insult as evidenced by a steady rise in total cellular glutathione accompanied by microtubule reassembly to their normal organization 5 hours after treatment. To evaluate the role of total cellular glutathione in modulating the 1chloro-2,4-dinitrobenzene-induced cytoskeletal perturbation, we used 1-chloro-2,4-dinitrobenzene and/or buthiomine sulfoximine, an effective irreversible inhibitor of glutathione synthesis, to manipulate cellular glutathione levels. Incubation of 3T3 cells with 2.5 M 1-chloro-2,4-dinitrobenzene and 250 M buthiomine sulfoximine for 5 hours resulted in a complete depletion of total cellular glutathione accompanied by essentially complete loss of microtubules and marked alterations in the density and distribution pattern of microfilaments. Buthionine sulfoximine enhanced markedly the extent and duration of cellular glutathione depletion and the severity of microtubule disruption of 3T3 cells over the level achieved by 1-chloro-2,4-dinitrobenzene treatment alone. Furthermore, buthiomine sulfoximine also prevented the restoration of cellular glutathione content and microtubule reassembly that normally were evident 5 hours after 1-chloro-2,4-dinitrobenzene treatment. Exposure of 3T3 cells to 50 M 2-cyclohexene-l-one, which depletes free glutathione by conjugation, resulted in a comAbbreviation BSO DL-buthiomine-S-R-sulfoximine - CDNB 1-chloro-2,4-dinitrobenzene - CHX 2-cyclohexene-l-one - GSH glutathione - GST glutathione-S-transferase - MAPS microtubule-associated proteins - MF microfilaments - MT microtubules.     

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.     

An analysis of seed development inPisum sativum V. Fluorescence triple staining for investigating cotyledon cell development

Summary A triple staining technique has been developed to investigate the relationship between the increase in DNA content and initiation of storage protein synthesis in pea cotyledon cells. Cells were separated by incubation with macerozyme providing a population comparable to conventional chromic acid techniques but with the advantage of retained immunogenicity. The staining technique combined indirect immunofluorescence using specific antibodies against the storage protein, vicilin, and the cytoskeletal protein, tubulin, with the DNA stain, 46-diamidino-2-phenylindole. Using the enzymically separated cells, the staining method allowed the visualisation of vicilin deposits and microtubules and the quantification of DNA by image analysis in thesame cells. The distribution of cellular DNA contents and storage protein content increased with the size of embryo. In small embryos a proportion of mitotic cells were seen to have increased amounts of DNA, though no spindle abnormalities were seen. Storage protein could be detected by immunofluorescence in individual cells much earlier than reported by previous workers but never in mitotic cells. The sensitivity of the immunofluoresence technique for detecting storage protein was determined as 0.5 pg per cell by estimating the vicilin production in whole pea embryos using an enzyme immunoassay.Abbreviations BSA bovine serum albumin - DAPI 46-diamidino-2-phenylindole - DMSO dimethyl sulphoxide - EGTA ethyleneglycolbis-N,N,N,N-tetraacetic acid - ELISA enzyme linked immunosorbent assay - FITC fluorescein isothiocyanate - ISIT intensified silicon integrated target - MTSB microtubule stabilising buffer - PIPES piperazine-N,N-bis[2-ethane-sulfonic acid] - PMSF phenylmethylsulphonyl floride - TBS Tris buffered saline     

Origin of kinetochore microtubules in Chinese hamster ovary cells

We have attempted to determine whether chromosomal microtubules arise by kinetochore nucleation or by attachment of pre-existing microtubules. The appearance of new microtubules was investigated in vivo on kinetochores to which microtubules had not previously been attached. The mitotic apparatus of Chinese hamster ovary cells was reconstructed in three dimensions from 0.25 m thick serial sections, and the location of chromosomes, kinetochore outer disks, centrioles, virus-like particles and microtubules determined. Central to the interpretation of these data is a synchronization scheme in which cells entered Colcemid arrest without forming mitotic microtubules. Cells were synchronized by the excess thymidine method and exposed to 0.3 g/ml Colcemid for 8 h. Electron microscopic examination showed that this Colcemid concentration eliminated all microtubules. Mitotic cells were collected by shaking off, and cell counts showed that over 95% of the cells were in interphase when treatment began and thus were arrested without the kinetochores having been previously attached to microtubules. Cells were then incubated in fresh medium and fixed for high voltage electron microscopy at intervals during recovery. — In early stages of recovery, short microtubules were observed near and in contact with kinetochores and surrounding centrioles. Microtubules were associated with kinetochores facing away from centrosomes and far from any centrosomal microtubules, and thus were not of centrosomal origin. At a later stage of recovery, long parallel bundles of microtubules, terminating in the kinetochore outer disk, extended from kinetochores both toward and away from centrosomes. Because microtubules had never been attached to kinetochores, the possibility that kinetochore microtubles were initiated by microtubule stubs resistant to Colcemid was eliminated. Therefore we conclude that mammalian kinetochores can initiate microtubules in vivo, thus serving as microtubule organizing centers for the mitotic spindle, and that formation of kinetochore-microtubule bundles is not dependent on centrosomal activity.     

The plant cell body: a cytoskeletal tool for cellular development and morphogenesis

Summary Certain aspects of cellular behaviour in relation to growth and development of plants can be understood in terms of the cell body concept proposed by Daniel Mazia in 1993. During the interphase of the mitotic cell cycle, the plant cell body is held to consist of a nucleus and a perinuclear microtubule-organizing centre from which microtubules radiate into the cytoplasm. During mitosis and cytokinesis in meristematic cells, and also during the period of growth in post-mitotic cells immediately beyond the meristem, the plant cell body undergoes various characteristic morphological transformations, many of which are proposed as being related to changing structural connections with the actin-based component of the cytoskeleton and with specialized, plasma-membrane-associated sites at the cell periphery. In post-mitotic cells, these transformations of the plant cell body coincide with, and probably provide conditions for, the various pathways of development which such cells follow. They are also responsible, for the acquisition of new cellular polarities. Events in which the plant cell body participates include the formation of a mitotic spindle, phragmoplast, and new cell division wall, the rearrangement of a diffuse type of cell wall growth into tip growth (as occurs, e.g., during the initiation and subsequent development of root hairs), and the growth and division that occurs in reactivated vacuolate cells. If more evidence can be marshalled in support of the existence and properties of the plant cell body, then this concept could prove useful in interpreting the cytological bases of a range of developmental events in plants.Abbreviations CMT cortical microtubule - EMT endoplasmic microtubule - ER endoplasmic reticulum - MF microfilament - MT microtubule - MTOC microtubule-organizing centre - PPB preprophase band (of microtubules) - QC quiescent centre - VSC vesicle supply centre     

The tubulin cytoskeleton and its sites of nucleation in hyphal tips ofAllomyces macrogynus

Summary The tubulin cytoskeleton in hyphal tip cells ofAllomyces macrogynus was detected with an -tubulin monoclonal antibody and analyzed with microscopic and immunoblot techniques. The -tubulin antibody identified a 52 kilodalton polypeptide band on immunoblots. Immunfluorescence data were collected from formaldehyde-and cryofixed hyphae. Both methods provided similar images of tubulin localization. However, cryofixation yielded more consistent labeling and did not require detergent extraction or cell-wall lytic treatments. Tubulin was primarily localized as microtubules observed in the peripheral and central cytoplasmic regions and in mitotic spindles. Cytoplasmic microtubules were oriented parallel to the cells' longitudinal axis, with central microtubules more often varied in their alignment, and emanated from a region in the hyphal apex resulting in an apical zone of bright fluorescence. A thin layer of microtubules appearing as bands of fluorescence encircled many nuclei. Discrete spots of fluorescence were also associated with nuclei. The MPM-2 antibody, which recognizes phosphorylated epitopes of several proteins that may be involved in the regulation of microtubule nucleation, stained centrosomes but not apical regions of hyphae. Nocodazole was used to depolymerize the microtubule network and reveal its regions of origin. A hocodazole concentration of 0.01 g/ ml (3.3× 10^–8M) provided a 70 to 75% inhibition of hyphal tip growth and was used throughout this study. The number of cells having an apical zone of fluorescence declined by 15 min of exposure. This zone was present in only a few cells after 60 min. After 30 min, the central cytoplasm consisted of small microtubule fragments and nuclear-associated spots. A small number of peripheral microtubules and nuclear-associated spots persisted throughout nocodazole treatments. Spindle microtubules were restored by 30 min after removal of nocodazole. This was followed by the reappearance of the apical zone of fluorescence and then by central and peripheral cytoplasmic microtubules. Apical fluorescence coincided with the presence of a Spitzenkörper. The results suggest that the Spitzenkörper and centrosome function as centers of microtubule nucleation and organization during hyphal tip growth in this fungus.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidino-2-phenylindole - DMSO dimethylsulfoxide - FITC fluorescein isothiocyanate - IB incubation buffer - LN₂ liquid nitrogen - LSCM laser scanning confocal microscopy - MTOCs microtubule-organizing centers - PBS phosphate buffered saline - PIPES 1,4-piperazinedietha-nesulfonic acid - PFB PIPES fixation buffer - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - SPB spindle pole body - TEM transmission electron microscopy - YpSs yeast extract-inorganic phosphate-soluble starch     

Re-organization of microtubules during preprophase band development inAdiantum protonemata

Summary Microtubule organization during preprophase band development was investigated using immunofluorescence microscopy in filamentous protonemal cells (approx. 600 m in length, 20 m in width) ofAdiantum capillus-veneris L. Protonemata pre-cultured under red light were transferred to continuous blue light or total darkness to induce synchronous cell division. Preprophase bands were found under both light conditions. In an early stage of development, the preprophase band which is transverse to the cell axis overlapped with an interphase cortical array of microtubules which is random or parallel to the cell axis. The interphase cortical array disappeared thereafter. While the width of the preprophase band became narrow during development under dark conditions, under blue light conditions it did not.Spatial and temporal aspects of the disappearance of the interphase cortical array of microtubules were also investigated. The interphase cortical array began to disappear at nearly the same time as the beginning of preprophase band formation. Under blue light, the disruption of cortical microtubules started at approx. 150 m from the tip (approx. 120 m from the nucleus), and spread toward the tip as far as the nuclear region and toward the base to an area approx. 300–400 m from the tip. Cortical microtubules remained in the basal part of the protonema. The pattern of disappearance between the tip and nucleus could not be determined. Under dark conditions, the pattern of the disappearance of cortical microtubules was somewhat different in many cells from that encountered with exposure to blue light. Microtubules first re-oriented from longitudinal to transverse, and then gradually disappeared. In some cells, the pattern of disappearance was similar to that observed under blue light.Abbreviations DAPI 4, 6-diamidino-2-phenylindole - ICM interphase cortical microtubules - PBS phosphate buffered saline - PPB preprophase band - MT microtubule     

Long link induction between the microtubules of the manchette in intermediate stages of spermiogenesis

Summary Links unusual for their length and variable morphology have been described between manchette microtubules in late stages of rat spermiogenesis. In earlier stages of rat spermiogenesis obvious links longer than 160 Å are rare, the majority being 80 or 120 Å in length. The most easily discernible geometric pattern in cross-sections of assemblies of manchette microtubules in intermediate stages of rat spermiogenesis is that of linear arrays sometimes resulting in long and irregularly folded chains of closely linked microtubules. Colcemid disrupts these arrays and is responsible for the formation of more complex geometric patterns. Six hours after drug administration the manchette is dramatically reduced in length. Sheet-like links of variable dimensions and >160 Å in length interconnect not only microtubules but C-type microtubules as well as other links. These links are similar in morphology to those found in later stages of rat spermiogenesis. It is suggested that the formation of these links may perhaps be dependent upon aspects of microtubule disassembly.Supported by a grant to E. A. MacKinnon by the Medical Research Council of Canada.     

Acetylated tubulin is found in all microtubule arrays of two species of pine

Summary The distribution of acetylated tubulin in microtubule arrays of conifer cells was investigated by immunofluorescence techniques with 6-11B-1, a monoclonal antibody specific for posttranslationally acetylated -tubulins. In methacrylate sections ofPinus radiata andPinus conforta root tip cells, acetylated tubulin was detected in mitotic spindles, phragmoplasts, and cortical microtubules. Furthermore, staining of isolated, intact cells ofP. radiata andP. contorta indicated that all microtubule structures, including preprophase bands, prophase, metaphase and anaphase spindles, and phragmoplasts, contained some acetylated tubulin, and that the intensity of staining with 6-11B-1 was variable. For example, preprophase bands were lightly labelled, kinetochore fibres of anaphase spindles and phragmoplasts were heavily stained, and metaphase spindles had a granular appearance suggesting discontinuous acetylation of their constituent microtubules. This first report of the presence of acetylated tubulin in conifer cells is in contrast to our results with two species of angiosperms where no acetylated tubulin was detected. The significance of this and the variability of the intensity of staining in conifer arrays is discussed in terms of microtubule dynamics.     

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

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

Ultrastructural effects of the herbicide chlorpropham (CIPC) in root tip cells of wheat

The present ultrastructural investigation on the effects of 50 M chlorpropham (previously called CIPC) on growing roots of wheat (Triticum aestivum (L.) Thell cv. Vergina) was undertaken to clarify the mechanism of a carbamate herbicide action in plant cells, since the wide range of responses of plant cells to carbamate herbicides is based mainly on immunofluorescence studies. Cells of control roots contained abundant microtubules both in interphase and mitotic arrays. In chlorpropham-treated roots, however, no microtubules could be detected at all, neither in dividing nor in differentiating cells. Cycling cells became binucleate, polyploid or contained incomplete cell walls, the result of inhibition of cytokinesis. In long-term drug treatments (24 h or more) the affected cells entered a new cycle, which, however, did not progress beyond mid-metaphase. The nuclei of binucleate cells initiated prophase synchronously. Small vacuoles and Golgi vesicles were trapped within the nucleoplasm of the multilobed nuclei. In roots recovering from 8 h chlorpropham treatment, cells continued to exhibit polyploid nuclei, intranuclear vacuoles and incomplete walls. Microtubules reappeared but they were sparse and lacked a definite orientation. Preprophase cells did not form normal preprophase bands of microtubules, while mitotic cells occasionally contained microtubules bound to chromosomes and converged to minipoles. It is concluded that chlorpropham disorganized directly microtubules in addition to irreversibly affecting microtubule organizing centres, which failed to further support microtubule arrays.     

Study of mitosis in root-tip cells ofTriticum turgidum treated with the DNA-intercalating agent ethidium bromide

Summary This work examines mitosis in root-tip cells ofTriticum turgidum treated with the RNA synthesis inhibitor ethidium bromide, using tubulin immunolabeling and electron microscopy. The following aberrations were observed in ethidium bromideaffected cells: (1) incomplete chromatin condensation and nuclear-envelope breakdown; (2) delay of preprophase microtubule band maturation; (3) preprophase microtubule band assembly in cells displaying an interphase appearance of the nucleus; (4) prevention of the prophase spindle formation, caused by inhibition of perinuclear microtubule (Mt) formation and/or inability of the perinuclear Mts to assume bipolarity; (5) organization of an atypical metaphase spindle which is unable to arrange the chromosomes on the equatorial plane; (6) formation of an atypical perinuclear metaphase spindle in cells in which nuclear-envelope breakdown has been almost completely inhibited; (7) inhibition of the anaphase spindle formation as well as of anaphase chromosome movement; (8) disorganization of the atypical mitotic spindle during transition from mitosis to cytokinesis. The observations favor the following hypotheses. Nucleation of prophase spindle Mts is related to the mechanism that causes nuclear-envelope breakdown. The mitotic poles lack Mtnucleating and -organizing properties, and their function does not account for prophase and metaphase spindle assembly. The organization of the prophase spindle is not a prerequisite for the formation of the metaphase spindle; the metaphase spindle seems to be formed de novo by Mts nucleated on the nuclear envelope and/or in the immediate vicinity of chromosomes.Abbreviations 5-AU 5-aminouracil - EB ethidium bromide - EM electron microscopy - k-Mt kinetochore microtubule - Mt microtubule - MTOC microtubule-organizing center - NE nuclear envelope - NEB nuclear-envelope breakdown - PPB preprophase band of microtubules     

Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: localization in the mitotic apparatus

In this study we have examined the immunocytochemical distribution of calmodulin during mitosis of higher plant endosperm cells. Spindle development in these cells occurs without centrioles. Instead, asterlike microtubule converging centers appear to be involved in establishing spindle polarity. By indirect immunofluorescence and immunogold staining methods with anti-calmodulin antibodies, we found endosperm calmodulin to be associated with the mitotic apparatus, particularly with asterlike and/or polar microtubule converging centers and kinetochore microtubules, in an immunocytochemical pattern distinct from that of tubulin. In addition, endosperm calmodulin and calcium showed analogous distribution profiles during mitosis. Previous reports have demonstrated that calmodulin is associated with the mitotic apparatus in animal cells. The present observation that calmodulin is also associated with the mitotic apparatus in acentriolar, higher plant endosperm cells suggests that some of the regulatory mechanisms involved in spindle formation, microtubule disassembly, and chromosome movement in plant cells may be similar to those in animal cells. However, unlike animal cell calmodulin, endosperm calmodulin appears to associate with kinetochore microtubules throughout mitosis, which suggests a specialized role for higher plant calmodulin in the regulation of kinetochore microtubule dynamics.     

A model of microtubule oscillations

Simulations of microtubule oscillations have been obtained by a kinetic model including nucleation of microtubules, elongation by addition of GTP-loaded tubulin dimers, disassembly into oligomers, and dissolution of oligomers followed by nucleotide exchange at the free dimers. Dynamic instability is described by the on and off rates for dimer association in the growth phase, the rate of rapid shortening, and the transition rates for catastrophe and rescue. The latter are assumed to be completely determined by the current state of the system (short cap hypothesis). Microtubule oscillations and normal polymerizations measured by time-resolved X-ray scattering were used to test the model. The model is able to produce oscillations without further assumptions. However, in order to obtain good fits to the experimental data one requires an additional mechanism which prevents rapid desynchronization of the microtubules. One of several possible mechanisms that will be discussed is the destabilization of microtubules by the products of disassembly.Abbreviations MT(s) microtubule(s) - G-MT/S-MT microtubule in the state of growth/shortening - GTP guanosine 5-triphosphate - GDP guanosine 5-diphosphate - TU · GDP/TU · GTP tubulin dimer with GDP/GTP bound to the exchangeable nucleotide binding site - MAP(s) microtubule-associated protein(s) - PC tubulin phosphocellulose-purified tubulin - PIPES piperazine-1,4-bis(2-ethane sulfonic acid) - DDT dithiothreitol - EGTA ethylene glycol-O,O-bis(2-amino ethyl ether)-N,N,N,N-tetraacetic acid     

Re-orientation of cortical F-actin is not necessary for wound-induced microtubule re-orientation and cell polarity establishment

Summary To assess the relative roles of cortical actin and microtubule re-orientation in the establishment of new cell polarity, we have examined the kinetics of cortical actin re-orientation around a wedge-shaped wound in pea roots. Cortical actin re-orients from a transverse alignment to an approximately longitudinal orientation between 5 and 24h after wounding, that is, after the re-alignment of microtubules, which is known to occur before 5h post-wounding. F-actin in root cortical cells does not appear to be necessary for the establishment of new cell polarity around wounds, since normal MT re-alignment, and new planes of cell division are still established around a wound in cytochalasin treated roots. The cytochalasin treatment appeared to totally disrupt cortical and cytoplasmic F-actin in cells of the root cortex. However, in the apparent absence of F-actin in these cells, the rate of wound-induced cell division, but not cell expansion, is slower, and we suggest that an effect on the phragmosomal actin is involved. Finally, we demonstrate that new cell polarity around a wound is not established if microtubules are disrupted by the herbicide oryzalin, but after re-establishment of these arrays following a wash-out of the drug, the typical new planes of cell expansion are observed. We conclude that microtubules play a critical role in establishing and maintaining cell polarity in this system, and that cortical F-actin has a minor and presently unclear function in these processes.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - DMSO dimethylsulphoxide - EGTA ethyleneglycol-bis-(-aminoethyleter)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MBS m-maleido-benzoyl N-hydroxysuccinimide ester - MSB microtubule stabilizing buffer - MT microtubule - PIPES 1,4-piperazine-dietha-nesulphonic acid - PPB pre-prophase band - Rh-ph rhodamine phalloidin     

Immature erythroid cells with novel morphology and cytoskeletal organization in adultXenopus

Summary Nucleated erythrocytes of non-mammalian vertebrates are a useful model system for studying the correlation between changes in cell shape and cytoskeletal organization during cellular morphogenesis. They are believed to transform from spheres to flattened discs to ellipsoids. Our previous work on developing erythroblasts suggested that pointed cells containing incomplete, pointed marginal bands (MBs) of microtubules might be intermediate stages in the larval axolotl. To test whether the occurrence of such pointed cells was characteristic of amphibian erythrogenesis, we have utilized phenylhydrazine (PHZ)-induced anemia in adultXenopus. In this system, circulating erythrocytes are destroyed and replaced by erythroblasts that differentiate in the blood, making them experimentally accessible. Thus, we followed the time-course of morphological and cytoskeletal changes in the new erythroid population during recovery. During days 7–9 post-PHZ, pointed cells did indeed begin to appear, as did spherical and discoidal cells. The percentage of pointed cells peaked at days 11–13 in different animals, subsequently declining as the percentage of elliptical cells increased. Since degenerating old erythrocytes were still present when pointed cells appeared, we tested directly whether pointed ones were old or new cells. Blood was removed via the dorsal tarsus vein, and the erythrocytes washed, fluorescently tagged, and re-injected. In different animals, 2–8% of circulating erythrocytes were labeled. Subsequent to induction of anemia in these frogs, time-course sampling showed that no pointed cells were labeled, identifying them as new cells. Use of propidium iodide revealed large nuclei and cytoplasmic staining indicative of immaturity, and video-enhanced phase contrast and anti-tubulin immunofluorescence showed that the pointed cells contained pointed MBs. The results show that pointed cells, containing incomplete, pointed MBs are a consistent feature of amphibian erythrogenesis. These cells may represent intermediate stages in the formation of elliptical erythrocytes.Abbreviations MB marginal band - MS membrane skeleton - PHZ phenylhydrazine     

Orientation of cortical microtubules correlates with cell shape and division direction

Summary Cortical microtubules in the epidermis of regeneratingGraptopetalum plants were examined by in situ immunofluorescence. Paradermal slices of tissue were prepared by a method that preserves microtubule arrays and also maintains cell junctions. To test the hypothesis that cortical microtubule arrays align perpendicular to the direction of organ growth, arrays were visualized and their orientation quantified. A majority of microtubules are in transverse orientation with respect to the organ axis early in shoot development when the growth habit is uniform. Later in development, when growth habit is non-uniform and the tissue is contoured, cortical microtubules are increasingly longitudinal and oblique in orientation. Microtubules show only a minor change in orientation at the site of greatest curvature, the transition zone of a developing leaf. To assess the role of the division plane on orientation of arrays, the pattern of microtubules was examined in individual cells of common shape. Cells derived from transverse divisions have predominately transverse cortical arrays, whereas cells derived from oblique and longitudinal divisions have non-transverse arrays. The results show that, regardless of the stage of development, microtubules orient with respect to cell shape and plane of division. The results suggest that cytoskeletal function is best considered in small domains of growth within an organ.Abbrevations DMSO dimethylsulfoxide - EGTA ethylene glycol-bis-(ß-aminoethyl ether)-N, N, N, N-tetra acetic acid - FITC fluorescein isothiocyanate - MTSB microtubule stabilizing buffer - PBS phosphate buffered saline     

The effects of microtubule and microfilament disrupting agents on cytoskeletal arrays and wall deposition in developing cotton fibers

Summary The effects of various cytoskeletal disrupting agents (cholchicine, oryzalin, trifluralin, taxol, cytochalasins B and D) on microtubules, microfilaments and wall microfibril deposition were monitored in developing cotton fibers, using immunocytochemical and fluorescence techniques. Treatment with 10^–4 M colchicine, 10^–6 M trifluralin or 10^–6 M oryzalin resulted in a reduction in the number of microtubules, however, the drug-stable microtubules still appear to influence wall deposition. Treatment with 10^–5 M taxol increased the numbers of microtubules present within 15 minutes of application. New microtubules were aligned parallel to the existing ones, however, some evidence of random arrays was observed. Microtubules stabilized with taxol appeared to function in wall organization but do not undergo normal re-orientations during development. Microtubule disrupting agent had no detectable affect on the microfilament population. Exposure to either 4×10^–5 M cytochalasin B or 2×10^–6M cytochalasin D resulted in a disruption of microfilaments and a re-organization of microtubule arrays. Treatment with either cytochalasin caused a premature shift in the orientation of microtubules in young fibers, whereas in older fibers the microtubule arrays became randomly organized. These observations indicate that microtubule populations during interphase are heterogeneous, differing at least in their susceptibility to disruption by depolymerizing agents. Changes in microtubule orientation (induced by cytochalasin) indicate that microfilaments may be involved in regulating microtubule orientation during development.