latrunculin-b and oryzalin

Cell fusion is a pivotal process in fertilization and multinucleate cell formation. A plant cell is ubiquitously surrounded by a hard cell wall, and very few cell fusions have been observed except for gamete fusions. We recently reported that the fertilized central cell (the endosperm) absorbs the persistent synergid, a highly differentiated cell necessary for pollen tube attraction. The synergid-endosperm fusion (SE fusion) appears to eliminate the persistent synergid from fertilized ovule in

Topics: Actins; Arabidopsis; Brefeldin A; Bridged Bicyclo Compounds, Heterocyclic; Cell Fusion; Cyclin-Dependent Kinases; Cycloheximide; Deoxyadenosines; Dinitrobenzenes; Endosperm; Fertilization; Gene Expression Regulation, Plant; Mitosis; Phenotype; Polymerization; Roscovitine; Sulfanilamides; Thiazolidines

Both endogenous plant proteins and viral movement proteins associate with microtubules to promote their movement through plasmodesmata. The association of viral movement proteins with microtubules facilitates the formation of virus-associated replication complexes, which are required for the amplification and subsequent spread of the virus. However, the role of microtubules in the intercellular movement of plant proteins is less clear. Here we show that the SHORT-ROOT (SHR) protein, which moves between cells in the root to regulate root radial patterning, interacts with a type-14 kinesin, KINESIN G (KinG). KinG is a calponin homology domain kinesin that directly interacts with the SHR-binding protein SIEL (SHR-INTERACING EMBRYONIC LETHAL) and localizes to both microtubules and actin. Since SIEL and SHR associate with endosomes, we suggest that KinG serves as a linker between SIEL, SHR, and the plant cytoskeleton. Loss of KinG function results in a decrease in the intercellular movement of SHR and an increase in the sensitivity of SHR movement to treatment with oryzalin. Examination of SHR and KinG localization and dynamics in live cells suggests that KinG is a nonmotile kinesin that promotes the pausing of SHR-associated endosomes. We suggest a model in which interaction of KinG with SHR allows for the formation of stable movement complexes that facilitate the cell-to-cell transport of SHR.

Topics: Actins; Arabidopsis; Arabidopsis Proteins; Bridged Bicyclo Compounds, Heterocyclic; Dinitrobenzenes; Endosomes; Extracellular Space; Intracellular Space; Kinesins; Meristem; Microtubules; Models, Biological; Mutation; Nicotiana; Plant Epidermis; Plant Leaves; Plant Roots; Protein Domains; Protein Transport; Species Specificity; Subcellular Fractions; Sulfanilamides; Thiazolidines; Transcription Factors

Xanthomonas campestris pv. vesicatoria type III-secreted effectors were screened for candidates influencing plant cell processes relevant to the formation and maintenance of stromules in Nicotiana benthamiana lower leaf epidermis. Transient expression of XopL, a unique type of E3 ubiquitin ligase, led to a nearly complete elimination of stromules and the relocation of plastids to the nucleus. Further characterization of XopL revealed that the E3 ligase activity is essential for the two plastid phenotypes. In contrast to the XopL wild type, a mutant XopL lacking E3 ligase activity specifically localized to microtubules. Interestingly, mutant XopL-labeled filaments frequently aligned with stromules, suggesting an important, yet unexplored, microtubule-stromule relationship. High time-resolution movies confirmed that microtubules provide a scaffold for stromule movement and contribute to stromule shape. Taken together, this study has defined two populations of stromules: microtubule-dependent stromules, which were found to move slower and persist longer, and microtubule-independent stromules, which move faster and are transient. Our results provide the basis for a new model of stromule dynamics including interactions with both actin and microtubules.

Topics: Actin Cytoskeleton; Bacterial Proteins; Bridged Bicyclo Compounds, Heterocyclic; Dinitrobenzenes; Green Fluorescent Proteins; Microtubules; Mutation; Nicotiana; Plant Cells; Plants, Genetically Modified; Plastids; Sulfanilamides; Thiazolidines; Ubiquitin-Protein Ligases; Xanthomonas campestris

Fluorescence recovery after photo-bleaching (FRAP) involves the irreversible bleaching of a fluorescent protein within a specific area of the cell using a high-intensity laser. The recovery of fluorescence represents the movement of new protein into this area and can therefore be used to investigate factors involved in this movement. Here we describe a FRAP method to investigate the effect of a range of pharmacological agents on the targeting of Tobacco mosaic virus movement protein to plasmodesmata.

Topics: Arabidopsis; Brefeldin A; Bridged Bicyclo Compounds, Heterocyclic; Cytochalasin B; Dinitrobenzenes; Fluorescence Recovery After Photobleaching; Gene Expression Regulation, Plant; Genetic Vectors; Green Fluorescent Proteins; Membrane Glycoproteins; Microfilament Proteins; Microscopy, Confocal; Nicotiana; Plants, Genetically Modified; Plasmodesmata; Protein Synthesis Inhibitors; Protein Transport; Receptors, Estrogen; Recombinant Fusion Proteins; Seedlings; Sulfanilamides; Thiazolidines; Tobacco Mosaic Virus; Tubulin; Tubulin Modulators

Dynamin-related proteins (DRPs) are large GTPases involved in a wide range of cellular membrane remodeling processes. In Arabidopsis thaliana, two paralogous land plant-specific type DRPs, DRP2A and DRP2B, are thought to participate in the regulation of post-Golgi trafficking. Here, we examined their molecular properties and functional relationships. qRT-PCR and GUS assays showed that DRP2A and DRP2B were expressed ubiquitously, although their expressions were strongest around root apical meristems and vascular bundles. Yeast two-hybrid, bi-molecular fluorescent complementation, and co-immunoprecipitation mass spectrometry analyses revealed that DRP2A and DRP2B interacted with each other. In observations with confocal laser scanning microscopy and variable incidence angle fluorescent microscopy, fluorescent fusions of DRP2A and DRP2B almost completely co-localized and were mainly localized to endocytic vesicle formation sites of the plasma membrane, clathrin-enriched trans-Golgi network and the cell plate in root epidermal cells. Treatments with wortmannin, an inhibitor of phosphatidylinositol 3-/4-kinases, latrunculin B, an inhibitor of actin polymerization, and oryzalin, an inhibitor of microtubule polymerization, increased the resident time of DRP2A and DRP2B on the plasma membrane. These results show that DRP2A and DRP2B function coordinately in multiple pathways of post-Golgi trafficking in phosphatidylinositol 3- or 4-kinase and cytoskeleton polymerization-dependent manners.

Topics: Actins; Androstadienes; Arabidopsis; Arabidopsis Proteins; Bridged Bicyclo Compounds, Heterocyclic; Cell Membrane; Cytoskeleton; Dinitrobenzenes; Dynamins; Enzyme Inhibitors; Fluorescent Dyes; Golgi Apparatus; GTP-Binding Proteins; Mass Spectrometry; Microscopy, Confocal; Microscopy, Fluorescence; Plant Roots; Protein Transport; Sulfanilamides; Thiazolidines; Two-Hybrid System Techniques; Wortmannin

Correct positioning of the division plane is a prerequisite for plant morphogenesis. The preprophase band (PPB) is a key intracellular structure of division site determination. PPB forms in G2 phase as a broad band of microtubules (MTs) that narrows in prophase and specializes few-micrometer-wide cortical belt region, named the cortical division zone (CDZ), in late prophase. The PPB comprises several molecules, some of which act as MT band organization and others remain in the CDZ marking the correct insertion of the cell plate in telophase. Ran GTPase-activating protein (RanGAP) is accumulated in the CDZ and forms a RanGAP band in prophase. However, little is known about when and how RanGAPs gather in the CDZ, and especially with regard to their relationships to MT band formation. Here, we examined the spatial and temporal distribution of RanGAPs and MTs in the preprophase of onion root tip cells using confocal laser scanning microscopy and showed that the RanGAP band appeared in mid-prophase as the width of MT band was reduced to nearly 7 µm. Treatments with cytoskeletal inhibitors for 15 min caused thinning or broadening of the MT band but had little effects on RanGAP band in mid-prophase and most of late prophase cells. Detailed image analyses of the spatial distribution of RanGAP band and MT band showed that the RanGAP band positioned slightly beneath the MT band in mid-prophase. These results raise a possibility that RanGAP behaves differently from MTs during their band formation.

Topics: Antibodies; Bridged Bicyclo Compounds, Heterocyclic; Dinitrobenzenes; GTPase-Activating Proteins; Meristem; Microtubules; Models, Biological; Onions; Plant Proteins; Prophase; Protein Transport; Spatio-Temporal Analysis; Sulfanilamides; Thiazolidines; Tubulin

A number of viral proteins from plant viruses, other than movement proteins, have been shown to traffic intracellularly along actin filaments and to be involved in viral infection. However, there has been no report that a viral capsid protein may traffic within a cell by utilizing the actin/endoplasmic reticulum (ER) network. We used Tomato spotted wilt tospovirus (TSWV) as a model virus to study the cell biological properties of a nucleocapsid (N) protein. We found that TSWV N protein was capable of forming highly motile cytoplasmic inclusions that moved along the ER and actin network. The disruption of actin filaments by latrunculin B, an actin-depolymerizing agent, almost stopped the intracellular movement of N inclusions, whereas treatment with a microtubule-depolymerizing reagent, oryzalin, did not. The over-expression of a myosin XI-K tail, functioning in a dominant-negative manner, completely halted the movement of N inclusions. Latrunculin B treatment strongly inhibited the formation of TSWV local lesions in Nicotiana tabacum cv Samsun NN and delayed systemic infection in N. benthamiana. Collectively, our findings provide the first evidence that the capsid protein of a plant virus has the novel property of intracellular trafficking. The findings add capsid protein as a new class of viral protein that traffics on the actin/ER system.

Topics: Actin Cytoskeleton; Actins; Bridged Bicyclo Compounds, Heterocyclic; Dimethyl Sulfoxide; Dinitrobenzenes; Endoplasmic Reticulum; Inclusion Bodies, Viral; Intracellular Membranes; Microtubules; Myosins; Nicotiana; Nucleocapsid Proteins; Plant Epidermis; Plant Leaves; Plant Proteins; Protein Transport; Solanum lycopersicum; Sulfanilamides; Thiazolidines; Tospovirus; Virion

Endoplasmic reticulum (ER) immunolabeling in developing stomatal complexes and in the intervening cells of the stomatal rows (ICSRs) of Zea mays revealed that the cortical-ER forms distinct aggregations lining locally expanding wall regions. The polarized subsidiary cell mother cells (SMCs), displayed a cortical-ER-patch lining the wall region shared with the inducing guard cell mother cell (GMC), which disorganized during mitosis. In dividing SMCs, ER persisted in the preprophase band region and was unequally distributed in the mitotic spindle poles. The subsidiary cells (SCs) formed initially an ER-patch lining the common wall with the GMC or the young guard cells and afterwards an ER-ring in the junction of the SC wall with the neighboring ones. Distinct ER aggregations lined the ICSR wall regions shared with the SCs. The cortical-ER aggregations in stomatal cells of Z. mays were co-localized with actin filament (AF) arrays but both were absent from the respective cells of Triticum turgidum, which follow a different morphogenetic pattern. Experimental evidence showed that the interphase ER aggregations are organized by the respective AF arrays, while the mitotic ER aggregations by microtubules. These results revealed that AF and ER demarcated "cortical cytoplasmic domains" are activated below the locally expanding stomatal cell wall regions, probably via a mechanosensing mechanism triggered by the locally stressed plasmalemma/cell wall continuum. The probable role(s) of the local ER aggregations are discussed.

Topics: Actin Cytoskeleton; Bridged Bicyclo Compounds, Heterocyclic; Butanols; Colchicine; Cytochalasins; Dinitrobenzenes; Endoplasmic Reticulum; Mitosis; Neomycin; Phospholipase D; Plant Stomata; Spindle Apparatus; Sulfanilamides; Thiazolidines; Triticum; Type C Phospholipases; Zea mays

Arctic and alpine plants like Oxyria digyna have to face enhanced environmental stress. This study compared leaves from Oxyria digyna collected in the Arctic at Svalbard (78 degrees N) and in the Austrian Alps (47 degrees N) at cellular, subcellular, and ultrastructural levels. Oxyria digyna plants collected in Svalbard had significantly thicker leaves than the samples collected in the Austrian Alps. This difference was generated by increased thickness of the palisade and spongy mesophyll layers in the arctic plants, while epidermal cells had no significant size differences between the two habitats. A characteristic feature of arctic, alpine, and cultivated samples was the occurrence of broad stroma-filled chloroplast protrusions, 2 - 5 microm broad and up to 5 microm long. Chloroplast protrusions were in close spatial contact with other organelles including mitochondria and microbodies. Mitochondria were also present in invaginations of the chloroplasts. A dense network of cortical microtubules found in the mesophyll cells suggested a potential role for microtubules in the formation and function of chloroplast protrusions. No direct interactions between microtubules and chloroplasts, however, were observed and disruption of the microtubule arrays with the anti-microtubule agent oryzalin at 5 - 10 microM did not alter the appearance or dynamics of chloroplast protrusions. These observations suggest that, in contrast to studies on stromule formation in Nicotiana, microtubules are not involved in the formation and morphology of chloroplast protrusions in Oxyria digyna. The actin microfilament-disrupting drug latrunculin B (5 - 10 microM for 2 h) arrested cytoplasmic streaming and altered the cytoplasmic integrity of mesophyll cells. However, at the ultrastructural level, stroma-containing, thylakoid-free areas were still visible, mostly at the concave sides of the chloroplasts. As chloroplast protrusions were frequently found to be mitochondria-associated in Oxyria digyna, a role in metabolite exchange is possible, which may contribute to an adaptation to alpine and arctic conditions.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Chloroplasts; Cryoelectron Microscopy; Cytoskeleton; Dinitrobenzenes; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Microtubules; Plant Leaves; Polygonaceae; Sulfanilamides; Thiazolidines

Cellular growth and movement require both the control of direction and the physical capacity to generate forces. In animal cells directional control and growth forces are generated by the polymerization of and traction between the elements of the cytoskeleton. Whether actual forces generated by the cytoskeleton play a role in plant cell growth is largely unknown as the interplay between turgor and cell wall is considered to be the predominant structural feature in plant cell morphogenesis. We investigated the mechano-structural role of the cytoskeleton in the invasive growth of pollen tubes. These cells elongate rapidly by tip growth and have the ability to penetrate the stigmatic and stylar tissues in order to drill their way to the ovule. We used agents interfering with cytoskeletal functioning, latrunculin B and oryzalin, in combination with mechanical in vitro assays. While microtubule degradation had no significant effect on the pollen tubes' capacity to invade a mechanical obstacle, latrunculin B decreased the pollen tubes' ability to elongate in stiffened growth medium and to penetrate an obstacle. On the other hand, the ability to maintain a certain growth direction in vitro was affected by the degradation of microtubules but not actin filaments. To find out whether both cytoskeletal elements share functions or interact we used both drugs in combination resulting in a dramatic synergistic response. Fluorescent labeling revealed that the integrity of the microtubule cytoskeleton depends on the presence of actin filaments. In contrast, actin filaments seemed independent of the configuration of microtubules.

Topics: Actin Cytoskeleton; Bridged Bicyclo Compounds, Heterocyclic; Dinitrobenzenes; Germination; Microtubules; Pollen Tube; Sulfanilamides; Thiazolidines

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

We used confocal microscopy and in vitro analyses to show that Nicotiana tabacum WLIM1, a LIM domain protein related to animal Cys-rich proteins, is a novel actin binding protein in plants. Green fluorescent protein (GFP)-tagged WLIM1 protein accumulated in the nucleus and cytoplasm of tobacco BY2 cells. It associated predominantly with actin cytoskeleton, as demonstrated by colabeling and treatment with actin-depolymerizing latrunculin B. High-speed cosedimentation assays revealed the ability of WLIM1 to bind directly to actin filaments with high affinity. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed a highly dynamic in vivo interaction of WLIM1-GFP with actin filaments. Expression of WLIM1-GFP in BY2 cells significantly delayed depolymerization of the actin cytoskeleton induced by latrunculin B treatment. WLIM1 also stabilized actin filaments in vitro. Importantly, expression of WLIM1-GFP in Nicotiana benthamiana leaves induces significant changes in actin cytoskeleton organization, specifically, fewer and thicker actin bundles than in control cells, suggesting that WLIM1 functions as an actin bundling protein. This hypothesis was confirmed by low-speed cosedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of WLIM1. Taken together, these data identify WLIM1 as a novel actin binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.

Topics: Actin Cytoskeleton; Actins; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Dinitrobenzenes; Escherichia coli; Green Fluorescent Proteins; Microfilament Proteins; Molecular Sequence Data; Nicotiana; Plant Leaves; Plant Proteins; Protein Structure, Tertiary; Recombinant Fusion Proteins; Sulfanilamides; Thiazoles; Thiazolidines; Tubulin Modulators

Double labeling of microtubules and actin filaments revealed that in prophase subsidiary mother cells of Zea mays a monopolar prophase microtubule "half-spindle" is formed, which lines the nuclear hemisphere distal to the inducing guard mother cell. The nuclear hemisphere proximal to the guard mother cell is lined by an F-actin cap, consisting of a cortical F-actin patch and actin filaments originating from it. The microtubules of the "half-spindle" decline from the nuclear surface and terminate to the preprophase microtubule band. After disintegration of the latter, a bipolar metaphase spindle is organized. The polar F-actin cap persists during mitosis and early cytokinesis, extending to the chromosomes and the subsidiary cell daughter nucleus. In oryzalin treated subsidiary mother cells the prophase nuclei move away from the polar site. Cytochalasin B and latrunculin-B block the polar migration of subsidiary mother cell nuclei, but do not affect those already settled to the polar position. The prophase nuclei of latrunculin-B treated subsidiary mother cells are globally surrounded by microtubules, while the division plane of latrunculin-B treated subsidiary mother cells is misaligned. The prophase nuclei of brick 1 mutant Zea mays subsidiary mother cells without F-actin patch are also globally surrounded by microtubules. The presented data show that the prophase microtubule "half-spindle"-preprophase band complex anchors the subsidiary mother cell nucleus to the polar cell site, while the polar F-actin cap stabilizes the one metaphase spindle pole proximal to the inducing guard mother cell.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Cell Movement; Cell Nucleus; Cytochalasin B; Cytoskeleton; Dinitrobenzenes; Microtubule-Associated Proteins; Microtubules; Plant Leaves; Plant Proteins; Prophase; Sulfanilamides; Thiazolidines; Triticum; Zea mays

Sulphorhodamine-labelled oligosaccharides of xyloglucan are incorporated into the cell wall of Arabidopsis and tobacco roots, and of cultured Nicotiana tabacum cells by the transglucosylase (XET) action of XTHs. In the cell wall of diffusely growing cells, the subcellular pattern of XET action revealed a 'fibrillar' pattern, different from the xyloglucan localization. The fibrillar fluorescence pattern had no net orientation in spherical cultured cells. It changed to transverse to the long axis when the cells started to elongate, a feature mirroring the rearrangements of cortical microtubules and the accompanying cellulose deposition. Interference with the polymerization of microtubules and with cellulose deposition inhibited this strong and 'fibrillar'-organized XET-action, whereas interference with actin-polymerization only decreased the intensity of enzyme action. Epidermal cells of a mutant with reduced cellulose synthesis also had low XET action. Root hairs (tip-growing cells) exhibited high XET-action over all their length, but lacked the specific parallel pattern. In both diffuse- and tip-growing cell types extraction of the incorporated fluorescent xyloglucans by a xyloglucan-specific endoglucanase reduced the fluorescence, but the 'fibrillar' appearance in diffuse growing cells was not eliminated. These results show that XTHs act on the xyloglucans attached to cellulose microfibrils. After incorporation of the fluorescent oligosaccharides, the xyloglucans decorate the cellulose microfibrils and become inaccessible to hydrolytic enzymes.

Topics: Arabidopsis; Bridged Bicyclo Compounds, Heterocyclic; Cell Size; Cell Wall; Dinitrobenzenes; Glucans; Glycosyltransferases; Microfibrils; Nicotiana; Nitriles; Plant Roots; Sulfanilamides; Thiazoles; Thiazolidines

This study investigates how microtubules and microfilaments control organelle motility within the tips of conifer pollen tubes. Organelles in the 30-microm-long clear zone at the tip of Picea abies (L.) Karst. (Pinaceae) pollen tubes move in a fountain pattern. Within the center of the tube, organelles move into the tip along clearly defined paths, move randomly at the apex, and then move away from the tip beneath the plasma membrane. This pattern coincides with microtubule and microfilament organization and is the opposite of the reverse fountain seen in angiosperm pollen tubes. Application of latrunculin B, which disrupts microfilaments, completely stops growth and reduces organelle motility to Brownian motion. The clear zone at the tip remains intact but fills with thin tubules of endoplasmic reticulum. Applications of amiprophosmethyl, propyzamide or oryzalin, which all disrupt microtubules, stop growth, alter organelle motility within the tip, and alter the organization of actin microfilaments. Amiprophosmethyl inhibits organelle streaming and collapses the clear zone of vesicles at the extreme tip together with the disruption of microfilaments leading into the tip, leaving the plasma membrane intact. Propyzamide and oryzalin cause the accumulation of membrane tubules or vacuoles in the tip that reverse direction and stream in a reverse fountain. The microtubule disruption caused by propyzamide and oryzalin also reorganizes microfilaments from a fibrillar network into pronounced bundles in the tip cytoplasm. We conclude that microtubules control the positioning of organelles into and within the tip and influence the direction of streaming by mediating microfilament organization.

Topics: Actin Cytoskeleton; Bridged Bicyclo Compounds, Heterocyclic; Cell Culture Techniques; Dinitrobenzenes; Endoplasmic Reticulum; Flowers; Microtubules; Organelles; Picea; Sulfanilamides; Thiazoles; Thiazolidines

The mechanism of cytokinesis was investigated during the first asymmetric division in fucoid zygotes. A plate of actin assembled midway between daughter nuclei where microtubules interdigitated and defined the cytokinetic plane. A membrane was then deposited in islands throughout the cytokinetic plane; the islands eventually fused into a continuous partition membrane and cell plate material was deposited in the intermembrane space. All of these structures matured from the center of the cell outward (centrifugal maturation). Pharmacological agents were used to investigate the roles of microtubules, actin, and secretion in cytokinesis. The findings indicate a mechanism of cytokinesis that may be unique to the brown algae.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Cell Division; Cytokinesis; Cytoskeleton; Dinitrobenzenes; Phaeophyceae; Secretory Vesicles; Sulfanilamides; Thiazoles; Thiazolidines; Zygote

Embryogenesis of flowering plants establishes a basic body plan with apical-basal, radial and bilateral patterns from the single-celled zygote. Arabidopsis embryogenesis exhibits a nearly invariant cell division pattern and therefore is an ideal system for studies of early plant development. However, plant embryos are difficult to access for experimental manipulation, as they develop deeply inside maternal tissues. Here we present a method for the culture of zygotic Arabidopsis embryos in vitro. The technique omits excision of the embryo by culturing the entire ovule, thus greatly facilitating the time and effort involved. It enables external manipulation of embryo development and culture from the earliest developmental stages up to maturity. Administration of various chemical treatments as well as the use of different molecular markers is demonstrated together with standard techniques for visualizing gene expression and protein localization in in vitro cultivated embryos. The presented set of techniques allows for so far unavailable molecular physiology approaches in the study of early plant development.

Topics: Arabidopsis; Biological Transport; Biomarkers; Brefeldin A; Bridged Bicyclo Compounds, Heterocyclic; Cell Culture Techniques; Cytoskeleton; Dinitrobenzenes; Germination; Monensin; Seeds; Sulfanilamides; Thiazoles; Thiazolidines

The actin cytoskeleton has been proposed to be a major player in plant gravitropism. However, understanding the role of actin in this process is far from complete. To address this problem, we conducted an analysis of the effect of Latrunculin B (Lat B), a potent actin-disrupting drug, on root gravitropism using various parameters that included detailed curvature kinetics, estimation of gravitropic sensitivity, and monitoring of curvature development after extended clinorotation. Lat B treatment resulted in a promotion of root curvature after a 90 degrees reorientation in three plant species tested. More significantly, the sensitivity of maize (Zea mays) roots to gravity was enhanced after actin disruption, as determined from a comparison of presentation time of Lat B-treated versus untreated roots. A short 10-min gravistimulus followed by extended rotation on a 1-rpm clinostat resulted in extensive gravitropic responses, manifested as curvature that often exceeded 90 degrees. Application of Lat B to the cap or elongation zone of maize roots resulted in the disruption of the actin cytoskeleton, which was confined to the area of localized Lat B application. Only roots with Lat B applied to the cap displayed the strong curvature responses after extended clinorotation. Our study demonstrates that disrupting the actin cytoskeleton in the cap leads to the persistence of a signal established by a previous gravistimulus. Therefore, actin could function in root gravitropism by providing a mechanism to regulate the proliferation of a gravitropic signal originating from the cap to allow the root to attain its correct orientation or set point angle.

Topics: Actins; Biological Transport; Bridged Bicyclo Compounds, Heterocyclic; Cytoskeleton; Dimethyl Sulfoxide; Dinitrobenzenes; Gravitropism; Gravity Sensing; Indoleacetic Acids; Kinetics; Plant Root Cap; Plant Roots; Signal Transduction; Sulfanilamides; Thiazoles; Thiazolidines; Time Factors; Zea mays

Root hair formation is induced when lettuce seedlings are transferred from liquid medium at pH 6.0 to fresh medium at pH 4.0. If seedlings are transferred to pH 6.0, no root hairs are formed. We investigated the role of microtubules in this low pH-induced root hair initiation in lettuce. At the hair-forming zone in root epidermal cells, microtubules were perpendicular to the longitudinal axis of the cell just after pre-culture. This arrangement became disordered as early as 5 min after transfer to pH 4.0, and became random by 30 min later. At pH 4.0, the randomization extended to the entire hair-forming zone of seedlings; at pH 6.0, however, randomization did not occur and transverse microtubules were maintained. When seedlings at pH 6.0 were treated with microtubule-depolymerizing drugs, root hairs were formed. In contrast, when a microtubule-stabilizing drug, taxol, was added to the medium, no root hairs formed, even at pH 4.0. These results suggest that the transverse cortical microtubules inhibit root hair formation, and that their destruction is necessary for initiation. Furthermore, the microfilament-disrupting drugs cytochalasin B and latrunculin B inhibited root hair initiation, suggesting that actin filaments are necessary for root hair initiation.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Cytochalasin B; Dinitrobenzenes; Lactuca; Microtubules; Paclitaxel; Plant Epidermis; Plant Roots; Sulfanilamides; Thiazoles; Thiazolidines

In Zea mays L., changes in orientation of stems are perceived by the pulvinal tissue, which responds to the stimulus by differential growth resulting in upward bending of the stem. Gravity is perceived in the bundle sheath cells, which contain amyloplasts that sediment to the new cell base when a change in the gravity vector occurs. The mechanism by which the mechanical signal is transduced into a physiological response is so far unknown for any gravity perceiving tissue. It is hypothesized that this involves interactions of amyloplasts with the plasma membrane and/or ER via cytoskeletal elements. To gain further insights into this process we monitored amyloplast movements in response to gravistimulation. In a pharmacological approach we investigated how the dynamics of plastid sedimentation are affected by actin and microtubule (MT) disrupting drugs. Dark grown caulonemal filaments of the moss Physcomitrella patens respond to gravity vector changes with a reorientation of tip growth away from the gravity vector. MT distributions in tip cells were monitored over time and MTs were seen to accumulate preferentially on the lower flank of the tip 30 min after a 90 degree turn. Using a self-referencing Ca2+ selective ion probe, we found that growing caulonemal filaments exhibit a Ca2+ influx at the apical dome, similar to that reported previously for other tip growing cells. However, in gravistimulated Physcomitrella filaments the region of Ca2+ influx is not confined to the apex, but extends about 60 micrometers along the upper side of the filament. Our results indicate an asymmetry in the Ca2+ flux pattern between the upper and side of the filament suggesting differential activation of Ca2+ permeable channels at the plasma membrane.

Topics: Actin Cytoskeleton; Bridged Bicyclo Compounds, Heterocyclic; Bryopsida; Calcium; Calcium Channels; Cytoskeleton; Darkness; Dinitrobenzenes; Gravitropism; Gravity Sensing; Herbicides; Marine Toxins; Microscopy, Confocal; Microtubules; Plant Shoots; Plastids; Pulvinus; Sulfanilamides; Thiazolidines; Tubulin Modulators; Zea mays

Plasmalemmal ionic currents from enzymatically isolated protoplasts of suspension-cultured tobacco 'Bright Yellow-2' cells were investigated by whole-cell patch-clamp techniques. In all protoplasts, delayed rectifier outward K(+) currents having sigmoidal activation kinetics, no inactivation, and very slow deactivation kinetics were activated by step depolarization. Tail current reversal potentials were close to equilibrium potential E(K) when external [K(+)] was either 6 or 60 mM. Several channel blockers, including external Ba(2+), niflumic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, inhibited this outward K(+) current. Among the monovalent cations tested (NH(4)(+), Rb(+), Li(+), Na(+)), only Rb(+) had appreciable permeation (P(Rb)/P(K) (=) 0.7). In addition, in 60 mM K(+) solutions, a hyperpolarization-activated, time-dependent, inwardly rectifying K(+) current was observed in most protoplasts. This inward current activated very slowly, did not inactivate, and deactivated quickly upon repolarization. The tail current reversal potential was very close to E(K), and other monovalent cations (NH(4)(+), Rb(+), Li(+), Na(+)) were not permeant. The inward current was blocked by external Ba(2+) and niflumic acid. External Cs(+) reversibly blocked the inward current without affecting the outward current. The amplitude of the inward rectifier K(+) current was generally small compared to the amplitude of the outward K(+) current in the same cell, although this was highly variable. Similar amplitudes for both currents occurred in only 4% of the protoplasts in control conditions. Microfilament-depolymerizing drugs shifted this proportion to about 12%, suggesting that microfilaments participate in the regulation of K(+) currents in tobacco 'Bright Yellow-2' cells.

Topics: Actin Cytoskeleton; Bridged Bicyclo Compounds, Heterocyclic; Cations, Monovalent; Cell Membrane; Cells, Cultured; Cytochalasin B; Dinitrobenzenes; Membrane Potentials; Nicotiana; Paclitaxel; Potassium; Potassium Channel Blockers; Potassium Channels, Voltage-Gated; Protoplasts; Rhodamines; Sulfanilamides; Thiazoles; Thiazolidines

The movement protein (MP) of cowpea mosaic virus (CPMV) forms tubules on infected protoplasts and through plasmodesmata in infected plants. In protoplasts the MP fused to GFP (MP-GFP) was shown to localize in peripheral punctate structures and in long tubular structures extending from the protoplast surface. Using cytoskeletal assembly inhibitors (latrunculin B and oryzalin) and an inhibitor of the secretory pathway (brefeldin A), targeting of the MP to the peripheral punctate structures was demonstrated not to be dependent on an intact cytoskeleton or functional secretion pathway. Furthermore it was shown that a disrupted cytoskeleton had no effect on tubule formation but that the addition of brefeldin A severely inhibited tubule formation. The results presented in this paper suggest a role for a plasma membrane host factor in tubule formation of plant viral MPs.

Topics: Brefeldin A; Bridged Bicyclo Compounds, Heterocyclic; Comovirus; Cytoskeleton; Dinitrobenzenes; Fabaceae; Golgi Apparatus; Green Fluorescent Proteins; Herbicides; Luminescent Proteins; Plant Viral Movement Proteins; Protoplasts; Secretory Vesicles; Sulfanilamides; Thiazoles; Thiazolidines; Transfection; Viral Proteins

Cell was deposition was investigated during morphogenesis in zygotes of Pelvetia compressa (J. Agardh) De Toni. Young zygotes are spherical and wall is deposited uniformly, but at germination (about 10 h after fertilization) wall deposition becomes localized to the apex of the tip-growing rhizoid. Wall deposition was investigated before and after the initiation of tip growth by disrupting cytoskeleton, secretion or cellulose deposition; effects on wall strength and structure were examined. All three were involved in generating wall strength in both spherical and tip-growing zygotes, but their relative importance were different at the two developmental stages. Much of the wall strength in young zygotes was dependent on F-actin, whereas cellulose and a sulfated component, probably a fucan (F2), were most important in tip growing zygotes. Some treatments had contrasting effects at the two developmental stages; for example, disruption of F-actin or inhibition of secretion weakened walls in spherical zygotes but strengthened those in tip-growing zygotes. Transmission electron microscopic analysis showed that most treatments that altered wall strength induced modifications of internal wall structure.

Topics: Actins; Brefeldin A; Bridged Bicyclo Compounds, Heterocyclic; Cell Cycle; Cell Polarity; Cell Wall; Cellulose; Cytoskeleton; Dimethyl Sulfoxide; Dinitrobenzenes; Fertilization; Golgi Apparatus; Herbicides; Morphogenesis; Osmotic Pressure; Phaeophyceae; Polysaccharides; Seawater; Sulfanilamides; Thiazoles; Thiazolidines; Zygote

The statoliths in Chara rhizoids are denser and more diamagnetic than the cytoplasm, therefore they can be displaced inside a living cell by a sufficiently strong high gradient magnetic field (HGMF). An experimental setup for intracellular magnetophoresis of statoliths was developed. The movement of statoliths and rhizoid growth was measured by video microscopy either under the influence of gravity or a HGMF equivalent to about 2 g. The contribution of the cytoskeleton to statolith motility was assayed before and after depolymerizing microtubules with oryzalin and F-actin with latrunculin B. Application of latrunculin caused immediate cessation of growth, clumping of statoliths, and application of HGMF resulted in higher displacement of statoliths. Oryzalin had no effect on the behavior of statoliths. The data indicate that magnetophoresis is a useful tool to study the gravisensing system and rheology of the Chara rhizoid.

Topics: Actins; Bridged Bicyclo Compounds, Heterocyclic; Chlorophyta; Cytoplasm; Cytoskeleton; Dinitrobenzenes; Dose-Response Relationship, Drug; Elasticity; Gravitation; Gravity Sensing; Herbicides; Hypergravity; Magnetics; Plastids; Sulfanilamides; Thiazoles; Thiazolidines; Viscosity

The single-cell trichomes in wild-type Arabidopsis are either unbranched or have two to five branches. Using transgenic Arabidopsis plants expressing a green fluorescent protein-microtubule-associated protein4 fusion protein, which decorates the microtubular cytoskeleton, we observed that during trichome branching, microtubules reorient with respect to the longitudinal growth axis. Considering branching to be a localized microtubule-dependent growth reorientation event, we investigated the effects of microtubule-interacting drugs on branch induction in trichomes. In unbranched trichomes of the mutant stichel, a change in growth directionality, closely simulating branch initiation, could be elicited by a short treatment with paclitaxel, a microtubule-stabilizing drug, but not with microtubule-disrupting drugs. The growth reorientation appeared to be linked to increased microtubule stabilization and to aster formation in the treated trichomes. Taxol-induced microtubule stabilization also led to the initiation of new branch points in the zwichel mutant of Arabidopsis, which is defective in a kinesin-like microtubule motor protein and possesses trichomes that are less branched. Our observations suggest that trichome cell branching in Arabidopsis might be mediated by transiently stabilized microtubular structures, which may form a component of a multiprotein complex required to reorient freshly polymerizing microtubules into new growth directions.

Topics: Actins; Arabidopsis; Arabidopsis Proteins; Benzamides; Biopolymers; Bridged Bicyclo Compounds, Heterocyclic; Calmodulin-Binding Proteins; Cell Division; Cell Size; Dinitrobenzenes; Genes, Plant; Microscopy, Confocal; Microscopy, Electron, Scanning; Microtubule-Associated Proteins; Microtubules; Multiprotein Complexes; Mutation; Paclitaxel; Phenotype; Plant Proteins; Plants, Genetically Modified; Recombinant Fusion Proteins; Sulfanilamides; Thiazoles; Thiazolidines

A high resolution ultraviolet (UV) bright-field microscope was used to analyse the formation of Hechtian strands and the Hechtian reticulation that remain attached to the cell wall after plasmolysis and deplasmolysis of onion inner epidermal cells. In real time video images, UV microscopy allowed a detailed investigation of the dynamic behaviour of the plasma membrane during the processes of osmotic water loss and uptake. Furthermore, the role of cytoskeletal elements as possible linkers of the plasma membrane to the cell wall was probed by application of cytoskeletal drugs during plasmolysis. Microtubules were depolymerized in oryzalin, and latrunculin B was used to destabilize actin microfilaments. The results showed no visible changes in the formation of the Hechtian reticulation or strands. Plasmolysis forms appeared to be normal, indicating stong membrane-to-wall attachments independent of cytoskeletal elements. During re-expansion of the protoplast in deplasmolysis, the plasma membrane incorporated Hechtian strands and subprotoplasts, fused with the Hechtian reticulation and finally realigned at the cell wall.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Carbocyanines; Cell Membrane; Dinitrobenzenes; Fluorescent Dyes; Microscopy, Ultraviolet; Onions; Osmotic Pressure; Plant Epidermis; Sulfanilamides; Thiazoles; Thiazolidines

Spatial and temporal changes in F-actin during polarity establishment in Pelvetia compressa zygotes were investigated using vital staining with rhodamine phalloidin (RP). F-actin was localized to a patch in the cortex of young zygotes. When unilateral light was applied to induce a growth axis (photopolarization) in a population of zygotes, the cortical F-actin patches localized at the shaded pole (rhizoid pole of growth axis). Treatments that prevented photopolarization prevented localization of F-actin patches to the shaded pole. When the direction of the light treatment was reversed, the previous growth axis was abandoned and a new axis was established in the opposite direction. The F-actin patch repositioned to the new rhizoid pole within minutes of light reversal, indicating that F-actin was an immediate marker of the nascent growth axis. Repositioning probably occurred by disassembly of the initial patch and reassembly of a new one. The patch grew in size as zygotes developed, eventually becoming a ring just prior to rhizoid outgrowth. The rhizoid emerged at the site of the F-actin ring and, following germination, the ring was located in the subapical zone of the elongating tip.

Topics: Actins; Brefeldin A; Bridged Bicyclo Compounds, Heterocyclic; Dinitrobenzenes; Eukaryota; Fertilization; Light; Phalloidine; Rhodamines; Saponins; Sulfanilamides; Thiazoles; Thiazolidines; Zygote

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

Diatoms are a group of unicellular microalgae that are encased in a highly ornamented siliceous cell wall, or frustule. Pennate diatoms have bilateral symmetry and many genera possess an elongated slit in the frustule called the raphe, a feature synonymous with their ability to adhere and glide over a substratum, a process little understood. We have used cytoskeleton-disrupting drugs to investigate the roles of actin, myosin, and microtubules in diatom gliding or motility. No effect on diatom gliding was observed using the cytochalasins, known actin inhibitors, or the microtubule-inhibitors oryzalin and nocodazole. The latrunculins are a new group of anti-actin drugs, and we show here that they are potent inhibitors of diatom gliding, resulting in the complete disassociation of the raphe-associated actin cables. The recovery of actin staining and motility following latrunculin treatment was extremely fast. Cells exposed to latrunculin for 12 h recovered full function and actin staining within 5 sec of the drug being removed, demonstrating that the molecular components required for this motility system are immediately available. Butanedione monoxime (BDM), a known myosin inhibitor, also reversibly inhibited diatom gliding in a manner similar to the latrunculins. This work provides evidence that diatom gliding is based on an actin/myosin motility system.

Topics: Actin Cytoskeleton; Actins; Bridged Bicyclo Compounds, Heterocyclic; Cytochalasins; Diacetyl; Diatoms; Dinitrobenzenes; Enzyme Inhibitors; Microtubules; Movement; Myosins; Nocodazole; Sulfanilamides; Thiazoles; Thiazolidines

The role of the cytoskeleton in the regulation of chloroplast motility and positioning has been investigated by studying: (1) structural relationship of actin microfilaments, microtubules, and chloroplasts in cryofixed and freeze-substituted leaf cells of Arabidopsis; and (2) the effects of anti-actin (Latrunculin B; LAT-B) and anti-microtubule (Oryzalin) drugs on intracellular distribution of chloroplasts. Immunolabeling of leaf cells with two plant-actin specific antibodies, which react equivalently with all the expressed Arabidopsis actins, revealed two arrangements of actin microfilaments: longitudinal arrays of thick actin bundles and randomly oriented thin actin filaments that extended from the bundles. Chloroplasts were either aligned along the actin bundles or closely associated with the fine filaments. Baskets of actin microfilaments were also observed around the chloroplasts. The leaf cells labeled with an anti-tubulin antibody showed dense transverse arrays of cortical microtubules that exhibited no apparent association with chloroplasts. The application of LAT-B severely disrupted actin filaments and their association with chloroplasts. In addition, LAT-B induced aberrant aggregation of chloroplasts in the mesophyll and bundle sheath cells. Double labeling of LAT-B treated cells with anti-actin and anti-tubulin antibodies revealed that the microtubules in these cells were unaffected. Moreover, depolymerization of microtubules with Oryzalin did not affect the distribution of chloroplasts. These results provide evidence for the involvement of actin, but not tubulin, in the movement and positioning of chloroplasts in leaf cells. We propose that using motor molecules, some chloroplasts migrate along the actin cables directly, while others are pulled along the cables by the fine actin filaments. The baskets of microfilaments may anchor the chloroplasts during streaming and allow control over proper three-dimensional orientation to light.

Topics: Actins; Amino Acid Sequence; Antibodies, Monoclonal; Antibody Specificity; Arabidopsis; Bridged Bicyclo Compounds, Heterocyclic; Chloroplasts; Dinitrobenzenes; Microscopy, Fluorescence; Microtubules; Molecular Sequence Data; Organelles; Plant Leaves; Protein Binding; Sequence Homology, Amino Acid; Sulfanilamides; Thiazoles; Thiazolidines

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