Relationship between the generative cell and vegetative nucleus in pollen tubes of Nicotiana tabacum

Summary Fluorescence microscopy was used to visualize microtubules (Mts) and chromatin in an effort to further clarify the relationship between the generative cell (GC) and vegetative nucleus (VN) in pollen tubes of tobacco. Prominent Mt bundles are present in one or more GC extensions that can be finger-like or lamellar in form. While the VN is positioned distal to the GC in most cases, it can also straddle the cell or lie proximal to it. In all cases, however, extensions embrace, penetrate or clasp the VN. GC Mts are reorganized during the formation of the mitotic apparatus, and cell extensions are fully or partially withdrawn. By telophase in many pollen tubes, the VN shifts to a more proximal position and appears to adhere to the region of the GC containing the phragmoplast. Application of oryzalin leads to the disorganization of Mts, changes in cell shape, including the loss or alteration of cell extensions, and separation of the GC and VN in some cases. However, the position and polarity of the VN is maintained in most pollen tubes. The results indicate that GC Mts and cell extensions play a role in the association with the VN. However, the relationship appears to be controlled by other factors as well. Attention should now be directed at potential interactions involving the VN envelope, vegetative plasma membrane, GC plasma membrane and extracellular matrix.Abbreviations GC Generative cell - MGU male germ unit - Mt microtubule - VN vegetative nucleus     

Red light imaging for programmed cell death visualization and quantification in plant–pathogen interactions

Studies on plant–pathogen interactions often involve monitoring disease symptoms or responses of the host plant to pathogen-derived immunogenic patterns, either visually or by staining the plant tissue. Both these methods have limitations with respect to resolution, reproducibility, and the ability to quantify the results. In this study we show that red light detection by the red fluorescent protein (RFP) channel of a multipurpose fluorescence imaging system that is probably available in many laboratories can be used to visualize plant tissue undergoing cell death. Red light emission is the result of chlorophyll fluorescence on thylakoid membrane disassembly during the development of a programmed cell death process. The activation of programmed cell death can occur during either a hypersensitive response to a biotrophic pathogen or an apoptotic cell death triggered by a necrotrophic pathogen. Quantifying the intensity of the red light signal enables the magnitude of programmed cell death to be evaluated and provides a readout of the plant immune response in a faster, safer, and nondestructive manner when compared to previously developed chemical staining methodologies. This application can be implemented to screen for differences in symptom severity in plant–pathogen interactions, and to visualize and quantify in a more sensitive and objective manner the intensity of the plant response on perception of a given immunological pattern. We illustrate the utility and versatility of the method using diverse immunogenic patterns and pathogens.     

Structure based drug design and in vitro metabolism study: Discovery of N-(4-methylthiophenyl)-N,2-dimethyl-cyclopenta[d]pyrimidine as a potent microtubule targeting agent

We report a series of tubulin targeting agents, some of which demonstrate potent antiproliferative activities. These analogs were designed to optimize the antiproliferative activity of 1 by varying the heteroatom substituent at the 4′-position, the basicity of the 4-position amino moiety, and conformational restriction. The potential metabolites of the active compounds were also synthesized. Some compounds demonstrated single digit nanomolar IC₅₀ values for antiproliferative effects in MDA-MB-435 melanoma cells. Particularly, the S-methyl analog 3 was more potent than 1 in MDA-MB-435 cells (IC₅₀?=?4.6?nM). Incubation of 3 with human liver microsomes showed that the primary metabolite of the S-methyl moiety of 3 was the methyl sulfinyl group, as in analog 5. This metabolite was equipotent with the lead compound 1 in MDA-MB-435 cells (IC₅₀?=?7.9?nM). Molecular modeling and electrostatic surface area were determined to explain the activities of the analogs. Most of the potent compounds overcome multiple mechanisms of drug resistance and compound 3 emerged as the lead compound for further SAR and preclinical development.     

The 3.5-Å CryoEM Structure of Nanodisc-Reconstituted Yeast Vacuolar ATPase Vo Proton Channel

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Interaction of auxin, light, and mechanical stress in orienting microtubules in relation to tropic curvature in the epidermis of maize coleoptiles

Summary Changes in the orientation of cortical microtubules (longitudinal vs. transverse with respect to the long cell axis) at the outer epidermal wall of maize coleoptile segments were induced by auxin, red or blue light, and mechanical stresses (cell extension or compression produced by bending). Immunofluorescent techniques were used for the quantitative determination of frequency distributions of microtubule orientation. Detailed kinetic studies showed that microtubule reorientations are temporally correlated with the simultaneously measured changes in growth rate elicited by auxin, red light, or blue light. Growth inhibition induced by depletion of endogenous auxin produces a longitudinal microtubule pattern that can be changed into a transverse pattern in a dose-dependent manner by applying exogenous auxin. A mid-point pattern with equal frequencies of longitudinal and transverse microtubules was adjusted at 2 mol/1 auxin. Bending stress applied under these conditions adjusts permanent, maximally longitudinal and transverse microtubule orientations at the compressed and extended segment sides, respectively, quantitatively mimicking the responses to differential flank growth during phototropic and gravitropic curvature. During tropic curvature the changes in microtubule pattern reflect the distribution of growth rather than the distribution of auxin. The microtubule pattern responds to auxin-dependent growth changes and mechanical stress in a synergistic manner, confirming the functional equivalence of these factors in affecting microtubule orientation. Similar results were obtained when segment growth was altered by blue or red light instead of auxin in the presence or absence of mechanical stress. It is concluded from these results that growth changes, elicited by auxin, light, etc., and mechanical stress affect microtubule orientation through a common signal perception and transduction chain.Abbreviations IAA indole-3-acetic acid (auxin) - MT cortical microtubule     

A mycorrhizal fungus changes microtubule orientation in tobacco root cells

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

Formation of a phragmosome-like structure in centrifuged protonemal cells of Adiantum capillus-veneris L.

A phragmosome (PS) is a transvacuolar aggregation of cytoplasm that develops in the plane of future cytokinesis and is found specifically in highly vacuolated cells. Although protonemal cells of Adiantum capillus-veneris L. usually do not form a PS, a PS-like structure developed at the site of a preprophase band (PPB) of microtubules (MTs) when the nucleus and endoplasm were displaced from the division site by centrifugation, leaving a PPB in the cortical cytoplasm. The PS-like structure contained endoplasmic MTs, F-actin, oil droplets and mitochondria. The structure did not develop when the cells were centrifuged before the formation of a PPB. Application of amiprophos-methyl (APM) before development of the PPB strongly inhibited the formation of the PS-like structure after centrifugation. The PS-like structure was dispersed after cytokinesis which occurred in the region of the displaced nucleus. Treatment with APM after the formation of the PS-like structure arrested the cell cycle at the M phase and inhibited the degradation of this structure. These results suggest that development of a PS-like structure is associated both with the formation of a PPB and with the stage of the cell cycle. Received: 9 July 1996 / Accepted: 12 September 1996     

Sites of microtubule reorganization in tobacco BY-2 cells during cell-cycle progression

Summary The sites of microtubule (MT) reorganization were examined in synchronized tobacco BY-2 cells. The MTs of these cells were completely destroyed by a combined cold and drug treatment at 0 °C with 100 M propyzamide for 3 h. After the cells were washed and cultured at 30 °C, the reorganization of MTs was observed in detail. Sites for MT reorganization at each stage of the cell cycle were identified on the cell cortex and nuclei, the mitotic apparatus, the nuclei (or the nuclei and cell cortex), and the cell cortex in the S-G₂ phase, M phase, M/G₁ interface, and g₁ phase, respectively. The polypeptide synthesis elongation factor (EF)-1 is co-localized with these sites of MT reorganization. At some stages, microfilaments (MFs) were found to be involved in the reorganization of MTs. Based on these results, the mode of MT reorganization during cell cycle progression is discussed.Abbreviations EF-1 elongation factor 1 - MAP microtubule-associated protein - MF microfilament - MIs mitotic indices - MT microtubule     

End-of-Life Infrastructure Economics for "Clean Vehicles" in the United States

Rising fuel prices and concern over emissions are prompting automakers and legislators to introduce and evaluate "clean vehicles" throughout the United States. Hybrid electric vehicles (HEVs) are now on the roads, electric vehicles (EVs) have been test marketed, and niche vehicles such as high-fuel-economy microcars are being considered for introduction. As these vehicles proliferate and mature, they will eventually reach their end of life (EOL). In the United States, an extensive recycling infrastructure exists for conventional, internal combustion engine (ICE) vehicles. Its primary constituents are the disassembler and the shredder. These industries, as well as battery recyclers, are expected to play integral roles in the EOL processing of clean vehicles.
A model of the automobile-recycling infrastructure and goal programming techniques are used to assess the materials streams and process profitabilities for several different clean vehicles. Two-seat EVs with lead-acid or NiMH batteries are compared with two- and four-seat HEVs and microcars. Changes to the nonferrous content in the vehicle bodies are explored and compared for the effect on processing profit-ability. Despite limitations associated with the linearity of goal programming techniques, application of this tool can still provide informative first-order results. Results indicate that although these clean vehicles may not garner the same profit levels as conventional ICE vehicles, they are profitable to process if there are markets for parts and if there are sufficient quantities of nonferrous materials.     

Fine-Structural Changes Associated with Pseudocyst Formation in Trichomitus batrachorum*

SYNOPSIS. Several fine-structural changes were observed during formation of the so-called pseudocysts of Trichomitus batrachorum grown in an agar-free medium. Among these changes the most frequent were those of the undulating membrane complex. Internalization of the marginal lamella combined with disappearance of the fin-like, membranous dorsal fold occurred with the highest frequency. In many organisms these alterations were accompanied by internalization of the recurrent flagellum, which, however, remained external in other, presumably earlier stages of pseudocyst formation. In some of the internalized and even still external recurrent flagella the microtubules were in various states of disassembly. The often distended flagellar membrane enclosed large accumulations of filaments 5 nm in diameter. Internalization of the anterior flagella also was noted. Changes similar to those characteristic of the recurrent flagella were not seen, however, among the microtubules of the anterior flagella or among those of the pelta or the axostyle.