cytochalasin-d and oryzalin

Intercellular protein movement is an important mechanism in plant development. Here we present an integrated protocol that utilizes an inducible system to block plasmodesmata-dependent movement and assessment of fluorescent recovery after photobleaching (FRAP) to identify compounds that influence intercellular protein movement.

Topics: Arabidopsis; Arabidopsis Proteins; Cytochalasin D; Dinitrobenzenes; Estradiol; Fluorescence Recovery After Photobleaching; Gene Expression Regulation, Plant; Green Fluorescent Proteins; Microscopy, Confocal; Microscopy, Fluorescence; Molecular Imaging; Plants, Genetically Modified; Plasmids; Plasmodesmata; Protein Transport; Recombinant Fusion Proteins; Seedlings; Sulfanilamides; Transcription Factors; Transgenes; Tubulin Modulators

Previous studies of protonemal morphogenesis in mosses have focused on the cytoskeletal basis of tip growth and the production of asexual propagules. This study provides the first comprehensive description of the differentiation of caulonemata and rhizoids, which share the same cytology, and the roles of the cytoskeleton in organelle shaping and spatial arrangement.. Light and electron microscope observations were carried out on in vitro cultured and wild protonemata from over 200 moss species. Oryzalin and cytochalasin D were used to investigate the role of the cytoskeleton in the cytological organization of fully differentiated protonemal cells; time-lapse photography was employed to monitor organelle positions.. The onset of differentiation in initially highly vacuolate subapical cells is marked by the appearance of tubular endoplasmic reticulum (ER) profiles with crystalline inclusions, closely followed by an increase in rough endoplasmic reticulum (RER). The tonoplast disintegrates and the original vacuole is replaced by a population of vesicles and small vacuoles originating de novo from RER. The cytoplasm then becomes distributed throughout the cell lumen, an event closely followed by the appearance of endoplasmic microtubules (MTs) in association with sheets of ER, stacks of vesicles that subsequently disperse, elongate mitochondria and chloroplasts and long tubular extensions at both poles of the nucleus. The production of large vesicles by previously inactive dictysomes coincides with the deposition of additional cell wall layers. At maturity, the numbers of endoplasmic microtubules decline, dictyosomes become inactive and the ER is predominantly smooth. Fully developed cells remain largely unaffected by cytochalasin; oryzalin elicits profound cytological changes. Both inhibitors elicit the formation of giant plastids. The plastids and other organelles in fully developed cells are largely stationary.. Differentiation of caulonemata and rhizoids involves a remarkable series of cytological changes, some of which closely recall major events in sieve element ontogeny in tracheophytes. The cytology of fully differentiated cells is remarkably similar to that of moss food-conducting cells and, in both, is dependent on an intact microtubule cytoskeleton. The disappearance of the major vacuolar apparatus is probably related to the function of caulonema and rhizoids in solute transport. Failure of fully differentiated caulonema and rhizoid cells to regenerate is attributed to a combination of endo-reduplication and irreversible tonoplast fragmentation. The formation of giant plastids, most likely by fusion, following both oryzalin and cytochalasin treatments, suggests key roles for both microtubules and microfilaments in the spatial arrangement and replication of plastids.

Topics: Bryophyta; Cell Differentiation; Cytochalasin D; Cytoskeleton; Dinitrobenzenes; Endoplasmic Reticulum; Microscopy, Electron, Transmission; Microscopy, Interference; Microtubules; Sulfanilamides; Tubulin Modulators

Elongation of diffusely expanding plant cells is thought to be mainly under the control of cortical microtubules. Drug treatments that disrupt actin microfilaments, however, can reduce elongation and induce radial swelling. To understand how microfilaments assist growth anisotropy, we explored their functional interactions with microtubules by measuring how microtubule disruption affects the sensitivity of cells to microfilament-targeted drugs. We assessed the sensitivity to actin-targeted drugs by measuring the lengths and diameters of expanding roots and by analysing microtubule and microfilament patterns in the temperature-sensitive Arabidopsis thaliana mutant microtubule organization 1 (mor1-1), along with other mutants that constitutively alter microtubule arrays. At the restrictive temperature of mor1-1, root expansion was hypersensitive to the microfilament-disrupting drugs latrunculin B and cytochalasin D, while immunofluorescence microscopy showed that low doses of latrunculin B exacerbated microtubule disruption. Root expansion studies also showed that the botero and spiral1 mutants were hypersensitive to latrunculin B. Hypersensitivity to actin-targeted drugs is a direct consequence of altered microtubule polymer status, demonstrating that cross-talk between microfilaments and microtubules is critical for regulating anisotropic cell expansion.

Topics: Actin Cytoskeleton; Actins; Arabidopsis; Arabidopsis Proteins; Bridged Bicyclo Compounds, Heterocyclic; Cytochalasin D; Dinitrobenzenes; Microtubule-Associated Proteins; Microtubules; Mutation; Paclitaxel; Plant Roots; Sulfanilamides; Thiazoles; Thiazolidines; Tubulin Modulators

Apicomplexan parasites divide and replicate through a complex process of internal budding. Daughter cells are preformed within the mother on a cytoskeletal scaffold, endowed with a set of organelles whereby in the final stages the mother disintegrates and is recycled in the emerging daughters. How the cytoskeleton and the various endomembrane systems interact in this dynamic process remains poorly understood at the molecular level. Through a random YFP fusion screen we have identified two Toxoplasma gondii proteins carrying multiple membrane occupation and recognition nexus (MORN) motifs. MORN1 is highly conserved among apicomplexans. MORN1 specifically localizes to ring structures at the apical and posterior end of the inner membrane complex and to the centrocone, a specialized nuclear structure that organizes the mitotic spindle. Time-lapse imaging of tagged MORN1 revealed that these structures are highly dynamic and appear to play a role in nuclear division and daughter cell budding. Overexpression of MORN1 resulted in severe but specific defects in nuclear segregation and daughter cell formation. We hypothesize that MORN1 functions as a linker protein between certain membrane regions and the parasite's cytoskeleton. Our initial biochemical analysis is consistent with this model. Whereas recombinant MORN1 produced in bacteria is soluble, in the parasite MORN1 was associated with the cytoskeleton after detergent extraction.

Topics: Amino Acid Sequence; Animals; Cell Division; Cell Membrane; Cytochalasin D; Cytokinesis; Cytoskeletal Proteins; Cytoskeleton; Dinitrobenzenes; Membrane Proteins; Mitosis; Molecular Sequence Data; Protozoan Proteins; Recombinant Proteins; Repetitive Sequences, Amino Acid; Spindle Apparatus; Sulfanilamides; Toxoplasma

Recently, three Chenopodiaceae species, Bienertia cycloptera, Bienertia sinuspersici, and Suaeda aralocaspica, were shown to possess novel C(4) photosynthesis mechanisms through the compartmentalization of organelles and photosynthetic enzymes into two distinct regions within a single chlorenchyma cell. Bienertia has peripheral and central compartments, whereas S. aralocaspica has distal and proximal compartments. This compartmentalization achieves the equivalent of spatial separation of Kranz anatomy, including dimorphic chloroplasts, but within a single cell. To characterize the mechanisms of organelle compartmentalization, the distribution of the major organelles relative to the cytoskeleton was examined. Examination of the distribution of the cytoskeleton using immunofluorescence studies and transient expression of green fluorescent protein-tagged cytoskeleton markers revealed a highly organized network of actin filaments and microtubules associating with the chloroplasts and showed that the two compartments in each cell had different cytoskeletal arrangements. Experiments using cytoskeleton-disrupting drugs showed in Bienertia and S. aralocaspica that microtubules are critical for the polarized positioning of chloroplasts and other organelles. Compartmentalization of the organelles in these species represents a unique system in higher plants and illustrates the degree of control the plant cell has over the organization and integration of multiorganellar processes within its cytoplasm.

Topics: Actin Cytoskeleton; Biomarkers; Carbon; Cell Compartmentation; Cell Polarity; Chenopodiaceae; Chloroplasts; Cytochalasin D; Cytoskeleton; Dinitrobenzenes; Green Fluorescent Proteins; Microtubules; Organelles; Photosynthesis; Sulfanilamides

Plant cells expand by exocytosis of wall material contained in Golgi-derived vesicles. We examined the role of local instability of the actin cytoskeleton in specifying the exocytosis site in Arabidopsis root hairs. During root hair growth, a specific actin cytoskeleton configuration is present in the cell's subapex, which consists of fine bundles of actin filaments that become more and more fine toward the apex, where they may be absent. Pulse application of low concentrations of the actin-depolymerizing drugs cytochalasin D and latrunculin A broadened growing root hair tips (i.e., they increased the area of cell expansion). Interestingly, recovery from cytochalasin D led to new growth in the original growth direction, whereas in the presence of oryzalin, a microtubule-depolymerizing drug, this direction was altered. Oryzalin alone, at the same concentration, had no influence on root hair elongation. These results represent an important step toward understanding the spatial and directional regulation of root hair growth.

Topics: Actins; Arabidopsis; Bridged Bicyclo Compounds, Heterocyclic; Cell Division; Cytochalasin D; Cytoskeleton; Dinitrobenzenes; Dose-Response Relationship, Drug; Microtubules; Nucleic Acid Synthesis Inhibitors; Plant Roots; Sulfanilamides; Thiazoles; Thiazolidines

Endoplasmic reticulum (ER) organization in the dividing cells of the pterophyte Asplenium nidus and of the gymnosperms Pinus brutia and Pinus nigra has been studied by immunolocalization techniques using the monoclonal antibody 2E7, which recognizes luminar ER resident proteins containing C-terminal HDEL sequences. In the pterophyte, the ER reorganization during cell cycle is similar to that in angiosperms. Among others, prominent ER gatherings were found at the mitotic spindle poles and in the phragmoplast during cytokinesis. However, in the gymnosperms examined, the ER displays a unique pattern of reorganization not described so far. In both the Pinus species, well-defined ER patterns are successively formed during cell cycle. They are the preprophase ER-band, the prophase- metaphase- and anaphase ER-spindle, the interzonal ER-system, the ER-phragmoplast and an ER-system lining the daughter cell wall. The ER patterns are closely similar to that of the correspondent microtubule (MT) arrangements with which they are co-organized. Observations made on P. nigra root-cells affected by oryzalin, colchicine and cytochalasin D favour the conclusion that the pattern of ER organization is controlled during mitosis and cytokinesis by the MT cytoskeleton.

Topics: Actin Cytoskeleton; Anaphase; Cell Division; Colchicine; Cytochalasin D; Dinitrobenzenes; Endoplasmic Reticulum; Ferns; Herbicides; Interphase; Metaphase; Microtubules; Nucleic Acid Synthesis Inhibitors; Pinus; Prophase; Sulfanilamides; Telophase

We found previously that in living cells of Oedogonium cardiacum and O. donnellii, mitosis is blocked by the drug cytochalasin D (CD). We now report on the staining observed in these spindles with fluorescently actin-labeling reagents, particularly Bodipy FL phallacidin. Normal mitotic cells exhibited spots of staining associated with chromosomes; frequently the spots appeared in pairs during prometaphase-metaphase. During later anaphase and telophase, the staining was confined to the region between chromosomes and poles. The texture of the staining appeared to be somewhat dispersed by CD treatment but it was still present, particularly after shorter (< 2 h) exposure. Electron microscopy of CD-treated cells revealed numerous spindle microtubules (MTs); many kinetochores had MTs associated with them, often laterally and some even terminating in the kinetochore as normal, but the usual bundle of kinetochore MTs was never present. As treatment with CD became prolonged, the kinetochores became shrunken and sunk into the chromosomes. These results support the possibility that actin is present in the kinetochore of Oedogonium spp. The previous observations on living cells suggest that it is a functional component of the kinetochore-MT complex involved in the correct attachment of chromosomes to the spindle.

Topics: Actins; Boron Compounds; Cell Nucleus; Chlorophyta; Cytochalasin D; Dinitrobenzenes; Fluorescent Dyes; Herbicides; Kinetochores; Microscopy, Fluorescence; Microtubules; Nucleic Acid Synthesis Inhibitors; Peptides, Cyclic; Spindle Apparatus; Sulfanilamides; Time Factors

We have used drugs to examine the role(s) of the actin and microtubule cytoskeletons in the intracellular growth and replication of the intracellular protozoan parasite, Toxoplasma gondii. By using a 5 minute infection period and adding the drugs shortly after entry we can treat parasites at the start of intracellular development and 6-8 hours prior to the onset of daughter cell budding. Using this approach we found, somewhat surprisingly, that reagents that perturb the actin cytoskeleton in different ways (cytochalasin D, latrunculin A and jasplakinolide) had little effect on parasite replication although they had the expected effects on the host cells. These actin inhibitors did, however, disrupt the orderly turnover of the mother cell organelles leading to the formation of a large residual body at the posterior end of each pair of budding parasites. Treating established parasite cultures with the actin inhibitors blocked ionophore-induced egression of tachyzoites from the host cells, demonstrating that intracellular parasites were susceptible to the effects of these inhibitors. In contrast, the anti-microtubule drugs oryzalin and taxol, and to a much lesser extent nocodazole, which affect microtubule dynamics in different ways, blocked parasite replication by disrupting the normal assembly of the apical conoid and the microtubule inner membrane complex (IMC) in the budding daughter parasites. Centrosome replication and assembly of intranuclear spindles, however, occurred normally. Thus, daughter cell budding per se is dependent primarily on the parasite microtubule system and does not require a dynamic actin cytoskeleton, although disruption of actin dynamics causes problems in the turnover of parasite organelles.

Topics: Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Calcimycin; Cell Division; Cells, Cultured; Cytochalasin D; Cytoskeleton; Dinitrobenzenes; Fibroblasts; Growth Inhibitors; Humans; Ionophores; Microscopy, Electron; Microtubules; Nocodazole; Organelles; Paclitaxel; Sulfanilamides; Thiazoles; Thiazolidines; Toxoplasma

While it is now recognised that transport within the endomembrane system may occur via membranous tubules, spatial regulation of this process is poorly understood. We have investigated the role of the cytoskeleton in regulating the motility and morphology of the motile vacuole system in hyphae of the fungus Pisolithus tinctorius by studying (1) the effects of anti-microtubule (oryzalin, nocodazole) and anti-actin drugs (cytochalasins, latrunculin) on vacuolar activity, monitored by fluorescence microscopy of living cells; and (2) the ultrastructural relationship of microtubules, actin microfilaments, and vacuoles in hyphae prepared by rapid-freezing and freeze-substitution. Anti-microtubule drugs reduced the tubular component of the vacuole system in a dose-dependent and reversible manner, the extent of which correlated strongly with the degree of disruption of the microtubule network (monitored by immunofluorescence microscopy). The highest doses of anti-microtubule drugs completely eliminated tubular vacuoles, and only spherical vacuoles were observed. In contrast, anti-actin drugs did not reduce the frequency of tubular vacuoles or the motility of these vacuoles, even though immunofluorescence microscopy confirmed perturbation of microfilament organisation. Electron microscopy showed that vacuoles were always accompanied by microtubules. Bundles of microtubules were found running in parallel along the length of tubular vacuoles and individual microtubules were often within one microtubule diameter of a vacuole membrane. Our results strongly support a role for microtubules, but not actin microfilaments, in the spatial regulation of vacuole motility and morphology in fungal hyphae.

Topics: Actin Cytoskeleton; Actins; Bridged Bicyclo Compounds, Heterocyclic; Cell Movement; Cryopreservation; Cytochalasin B; Cytochalasin D; Dinitrobenzenes; Dose-Response Relationship, Drug; Freeze Substitution; Fungi; Microscopy, Electron; Microscopy, Fluorescence; Microtubules; Nocodazole; Sulfanilamides; Thiazoles; Thiazolidines; Vacuoles

During the establishment of polarity, fucoid algal zygotes adhere to the substratum and select a growth axis according to environmental cues. Since little is known about the early events leading to axis selection, we investigated the chronology of cell adhesion, adhesive deposition, and axis selection induced by light (photopolarization). The requirements for secretion and the cytoskeleton in these processes and in the process of changing the orientation of an axis in response to new environmental cues (axis realignment) were also tested. Adhesive deposition occurred in two distinct stages: it was deposited uniformally on young zygotes (uniform primary adhesive) and later was deposited asymmetrically (polar secondary adhesive). Uniform primary adhesive deposition, cell adhesion, and photopolarization occurred simultaneously, and shortly thereafter, polar secondary adhesive deposition occurred at the future growth site. Uniform primary adhesive deposition and cell adhesion required secretion, but were independent of filamentous-actin (F-actin) and microtubule function. Photopolarization of young zygotes and polar secondary adhesive deposition required secretion but not microtubules. F-actin served to localize secondary adhesive deposition at the rhizoid pole; its function in polarization was more complex. F-actin was required for axis selection; however, its role in realignment of an axis depended on the light regime. The differing requirements for F-actin during development indicates that the axis is not static, but changes with time. These findings indicate that previous and future work on "axis formation" must be interpreted in the context of the developmental stage of the zygote.

Topics: Actins; Anti-Bacterial Agents; Bodily Secretions; Brefeldin A; Bridged Bicyclo Compounds, Heterocyclic; Cell Adhesion; Cyclopentanes; Cytochalasin D; Cytoskeleton; Dinitrobenzenes; Eukaryota; Light; Macrolides; Microscopy, Confocal; Monensin; Sulfanilamides; Thiazoles; Thiazolidines; Zygote

Topics: Actin Cytoskeleton; Actins; Carbamates; Cell Polarity; Colchicine; Cotyledon; Cytochalasin D; Cytoskeleton; Dinitrobenzenes; Gravitropism; Gravity Sensing; Herbicides; Microtubules; Mutation; Nucleic Acid Synthesis Inhibitors; Oryza; Paclitaxel; Phenylcarbamates; Plastids; Sulfanilamides; Tubulin; Urethane

The F-actin distribution in caulonemal tip cells of the moss Ceratodon purpureus was examined by rhodamine-phalloidin staining. Gravitropically-growing caulonemal tip cells of the moss possess a distinct alignment of microfilaments (MFs) in their apices. Axially oriented actin bundles run from subapical regions to the apex where they converge towards a central area of the tip, although bundles are absent from the central area itself thus forming a collar-like structure. During a unilateral red light irradiation the actin strands of the apical dome become reoriented towards the irradiated apical flank and still surround an area free of MFs, the point of prospective outgrowth. This process is closely correlated with the morphological effect of bulging and precedes the light-directed outgrowth. The collar structure is essential for the tubular growth form. In darkness, under the influence of antimicrotubule agents the structure is decomposed, the actin strands drift along the cell flanks and finally accumulate in randomly distributed areas where further growth takes place. The microtubules (MTs) are not involved in the phytochrome-mediated reorientation of the microfilaments. Unilateral red light suppresses the distorting effect of antimicrotubule drugs and restores the collar structure with a pronounced light-directed orientation. Instead, the MTs seem to be responsible for restricting the reorientation to the cell tip. This notion is based on the observation that the small area in the apical dome, which is normally the exclusive location of the light-regulated MF rearrangement, extends towards the cell base when MT inhibitors are applied before the unilateral red light irradiation. This in turn leads to a non-tubular expansion of the light-directed cell flank.

Topics: Actin Cytoskeleton; Actins; Bryopsida; Cell Polarity; Colchicine; Cytochalasin D; Cytoskeleton; Dinitrobenzenes; Herbicides; Light; Microscopy, Fluorescence; Microtubules; Phototropism; Sulfanilamides

The arrangement of the microtubule cytoskeleton in tip-growing and gravisensing Chara rhizoids has been documented by immunofluorescence microscopy. Predominantly axially oriented undulating bundles of cortical microtubules were found in the basal zone of the rhizoids and colocalized with the microfilament bundles underlying the cytoplasmic streaming. Microtubules penetrate the subapical zone, forming a three-dimensional network that envelops the nucleus and organelles. Microtubules are present up to 5 to 10 microns basal from the apical cytoplasmic region containing the statoliths. No microtubules were found in the apical zone of the rhizoid which is the site of tip growth and gravitropism. Depolymerization of microtubules by application of oryzalin does not affect cytoplasmic streaming and gravitropic growth until the relatively stationary and polarly organized apical and subapical cytoplasm is converted into streaming cytoplasm. When the statoliths and the apical cytoplasm are included in the cytoplasmic streaming, tip growth and gravitropism are stopped. Oryzalin-induced disruption of the microtubule cytoskeleton also results in a rearrangement of the dense network of apical and subapical microfilaments into thicker bundles, whereas disruption of the microfilament cytoskeleton by cytochalasin D had no effect on the organization of the microtubule cytoskeleton. It is, therefore, concluded that the arrangement of microtubules is essential for the polar cytoplasmic zonation and the functionally polar organization of the actin cytoskeleton which is responsible for the motile processes in rhizoids. Microtubules are not involved in the primary events of gravitropism in Chara rhizoids.

Topics: Chlorophyta; Cytochalasin D; Dinitrobenzenes; Gravitation; Microtubules; Sulfanilamides; Tubulin

Zoospores of Phytophthora cinnamomi are formed by cleavage of a multinucleate sporangium and contain nine different components that are distributed or oriented about a well-defined axis running through a pair of basal bodies near the nucleus. In this study, the importance of the cytoskeleton in establishing and maintaining cellular polarity was examined by using the anti-microtubule drug oryzalin and the anti-microfilament drug cytochalasin D (CD). The effects of the drugs on uncleaved and cleaving sporangia were determined, using fluorescence microscopy, for six of the components that are polarized in untreated cleaved cells: an astral microtubule (MT) array, the nucleus, mitochondria and three different types of vesicles, two of which are involved in directed exocytosis. CD had no effect upon the MT arrays, the positioning of nuclei or the polarized redistribution of mitochondria and vesicles to the cortical cytoplasm, although it did cause abnormal cleavage. The effects of oryzalin, however, indicate that the asymmetric disposition of the MT array is fundamental to zoospore polarities: when the array is itself eliminated with this drug, none of the other five elements show any signs of polar positioning within the cleaved sporangium. Oryzalin also caused abnormal cleavage similar to that seen in CD-treated cells. Most intriguing, however, was the finding that although the three vesicle types in cleaved, oryzalin-treated sporangia did not exhibit the polarized distribution seen in control and CD-treated cells, in many cases the vesicles had, nevertheless, lost their initially random distributions and had become concentrated in the cytoplasm adjacent to the abnormal cleavage planes. Thus although an intact MT array is required for segregation of the vesicles within the cortex, their redistribution to the cortex can somehow occur in the absence of MTs and actin microfilaments.

Topics: Actin Cytoskeleton; Cell Polarity; Cytochalasin D; Dinitrobenzenes; Microtubules; Phytophthora; Spores, Fungal; Sulfanilamides