The effects of microcystin-LR in Oryza sativa root cells: F-actin as a new target of cyanobacterial toxicity.

Microcystins are toxins produced by cyanobacteria, notorious for negatively affecting a wide range of living organisms, among which several plant species. Although microtubules are a well-established target of microcystin toxicity, its effect on filamentous actin (F-actin) in plant cells has not yet been studied. Τhe effects of microcystin-LR (MC-LR) and an extract of a microcystin-producing freshwater cyanobacterial strain (Microcystis flos-aquae TAU-MAC 1510) on the cytoskeleton (F-actin and microtubules) of Oryza sativa (rice) root cells were studied with light, confocal, and transmission electron microscopy. Considering the role of F-actin in endomembrane system distribution, the endoplasmic reticulum and the Golgi apparatus in extract-treated cells were also examined. F-actin in both MC-LR- and extract-treated meristematic and differentiating root cells exhibited time-dependent alterations, ranging from disorientation and bundling to the formation of ring-like structures, eventually resulting in a collapse of the F-actin network after longer treatments. Disorganization and eventual depolymerization of microtubules, as well as abnormal chromatin condensation were observed following treatment with the extract, effects which could be attributed to microcystins and other bioactive compounds. Moreover, cell cycle progression was inhibited in extract-treated roots, specifically affecting the mitotic events. As a consequence of F-actin network disorganization, endoplasmic reticulum elements appeared stacked and diminished, while Golgi dictyosomes appeared aggregated. These results support that F-actin is a prominent target of MC-LR, both in pure form and as an extract ingredient. Endomembrane system alterations can also be attributed to the effects of cyanobacterial bioactive compounds (including microcystins) on the F-actin cytoskeleton.

The intracellular and intercellular cross-talk during subsidiary cell formation in Zea mays: existing and novel components orchestrating cell polarization and asymmetric division.

Background: Formation of stomatal complexes in Poaceae is the outcome of three asymmetric and one symmetric cell division occurring in particular leaf protodermal cells. In this definite sequence of cell division events, the generation of subsidiary cells is of particular importance and constitutes an attractive model for studying local intercellular stimulation. In brief, an induction stimulus emitted by the guard cell mother cells (GMCs) triggers a series of polarization events in their laterally adjacent protodermal cells. This signal determines the fate of the latter cells, forcing them to divide asymmetrically and become committed to subsidiary cell mother cells (SMCs). Scope: This article summarizes old and recent structural and molecular data mostly derived from Zea mays, focusing on the interplay between GMCs and SMCs, and on the unique polarization sequence occurring in both cell types. Recent evidence suggests that auxin operates as an inducer of SMC polarization/asymmetric division. The intercellular auxin transport is facilitated by the distribution of a specific transmembrane auxin carrier and requires reactive oxygen species (ROS). Interestingly, the local differentiation of the common cell wall between SMCs and GMCs is one of the earliest features of SMC polarization. Leucine-rich repeat receptor-like kinases, Rho-like plant GTPases as well as the SCAR/WAVE regulatory complex also participate in the perception of the morphogenetic stimulus and have been implicated in certain polarization events in SMCs. Moreover, the transduction of the auxin signal and its function are assisted by phosphatidylinositol-3-kinase and the products of the catalytic activity of phospholipases C and D. Conclusion: In the present review, the possible role(s) of each of the components in SMC polarization and asymmetric division are discussed, and an overall perspective on the mechanisms beyond these phenomena is provided.

Local differentiation of cell wall matrix polysaccharides in sinuous pavement cells: its possible involvement in the flexibility of cell shape.

The distribution of homogalacturonans (HGAs) displaying different degrees of esterification as well as of callose was examined in cell walls of mature pavement cells in two angiosperm and two fern species. We investigated whether local cell wall matrix differentiation may enable pavement cells to respond to mechanical tension forces by transiently altering their shape. HGA epitopes, identified with 2F4, JIM5 and JIM7 antibodies, and callose were immunolocalised in hand-made or semithin leaf sections. Callose was also stained with aniline blue. The structure of pavement cells was studied with light and transmission electron microscopy (TEM). In all species examined, pavement cells displayed wavy anticlinal cell walls, but the waviness pattern differed between angiosperms and ferns. The angiosperm pavement cells were tightly interconnected throughout their whole depth, while in ferns they were interconnected only close to the external periclinal cell wall and intercellular spaces were developed between them close to the mesophyll. Although the HGA epitopes examined were located along the whole cell wall surface, the 2F4- and JIM5- epitopes were especially localised at cell lobe tips. In fern pavement cells, the contact sites were impregnated with callose and JIM5-HGA epitopes. When tension forces were applied on leaf regions, the pavement cells elongated along the stretching axis, due to a decrease in waviness of anticlinal cell walls. After removal of tension forces, the original cell shape was resumed. The presented data support that HGA epitopes make the anticlinal pavement cell walls flexible, in order to reversibly alter their shape. Furthermore, callose seems to offer stability to cell contacts between pavement cells, as already suggested in photosynthetic mesophyll cells.

Myelin Sheath Development in the Maxillary Nerve of the Newborn Pig.

Myelination, the ensheathing of neuronal axons by myelin, is important for the proper function of both central and peripheral nervous systems. Various studies have investigated the quantitative parameters of myelination in certain species. Pigs are among the species of which their use as laboratory animals in neuroscience research increased the past few decades. However, there is limited data regarding the myelination process in the pig. Moreover, the maxillary nerve is crucial for Pseudorabies Virus (PrV) neuropathogenesis. In this context, a quantitative analysis of various myelination parameters of the maxillary nerve was performed, during the first 5 weeks of porcine post-natal development, the time period, which exhibits the highest interest for PrV neuropathogenesis. The evaluation was conducted in four groups of uninfected pigs, at the time of birth (group 0w), at the age of 1 week (group 1w), 3 weeks (group 3w) and 5 weeks (group 5w), using toluidine blue staining, immunofluorescence and electron microscopy. Axon and fibre diameter, perimeter and surface, myelin sheath thickness and g-ratio were measured on histological sections transverse to the longitudinal axis of the maxillary nerve. The thickness of myelin sheath was 0.76 µm for group 0w, 0.94 µm for group 1w, 0.98 µm for group 3w and 1.03 µm for group 5w. The g-ratio was 0.529, 0.540, 0.542 and 0.531 for the respective animal groups. The results of this study contribute to the understanding of the myelination process in the pig will be used for the study of PrV effects on the myelination development of newborn piglets' maxillary nerve and may shed new light to their vulnerability to the virus.

Cell wall matrix polysaccharide distribution and cortical microtubule organization: two factors controlling mesophyll cell morphogenesis in land plants.

BACKGROUND AND AIMS: This work investigates the involvement of local differentiation of cell wall matrix polysaccharides and the role of microtubules in the morphogenesis of mesophyll cells (MCs) of three types (lobed, branched and palisade) in the dicotyledon Vigna sinensis and the fern Asplenium nidus. METHODS: Homogalacturonan (HGA) epitopes recognized by the 2F4, JIM5 and JIM7 antibodies and callose were immunolocalized in hand-made leaf sections. Callose was also stained with aniline blue. We studied microtubule organization by tubulin immunofluorescence and transmission electron microscopy. RESULTS: In both plants, the matrix cell wall polysaccharide distribution underwent definite changes during MC differentiation. Callose constantly defined the sites of MC contacts. The 2F4 HGA epitope in V. sinensis first appeared in MC contacts but gradually moved towards the cell wall regions facing the intercellular spaces, while in A. nidus it was initially localized at the cell walls delimiting the intercellular spaces, but finally shifted to MC contacts. In V. sinensis, the JIM5 and JIM7 HGA epitopes initially marked the cell walls delimiting the intercellular spaces and gradually shifted in MC contacts, while in A. nidus they constantly enriched MC contacts. In all MC types examined, the cortical microtubules played a crucial role in their morphogenesis. In particular, in palisade MCs, cortical microtubule helices, by controlling cellulose microfibril orientation, forced these MCs to acquire a truncated cone-like shape. Unexpectedly in V. sinensis, the differentiation of colchicine-affected MCs deviated completely, since they developed a cell wall ingrowth labyrinth, becoming transfer-like cells. CONCLUSIONS: The results of this work and previous studies on Zea mays (Giannoutsou et al., Annals of Botany 2013; 112: : 1067-1081) revealed highly controlled local cell wall matrix differentiation in MCs of species belonging to different plant groups. This, in coordination with microtubule-dependent cellulose microfibril alignment, spatially controlled cell wall expansion, allowing MCs to acquire their particular shape.

Three-dimensional versus two-dimensional ultrasound for fetal nasal bone evaluation in the second trimester.

OBJECTIVES: To compare two-dimensional with three-dimensional ultrasound evaluation of the fetal nasal bone in the second trimester. METHODS: A prospective, non-interventional study was conducted, in 55 singleton fetuses, between 18 and 24 weeks' gestation. Fetal nasal bone length was measured in the midsagittal plane by two-dimensional imaging and in the midsagittal and coronal plane with three-dimensional ultrasound. All three measurements were compared with one another using one-way repeated samples-measures ANOVA and paired samples t-test. RESULTS: The average fetal nasal bone length (mean ± SD) as determined by the three methods was 7.01 ± 0.94 mm for the two-dimensional midsagittal, 6.96 ± 1.34 mm for the three-dimensional midsagittal, and 6.98 ± 1.32 mm for the three-dimensional coronal plane; comparisons between one another were not statistically significant. Unilateral hypoplasia and bifid shape of the fetal nasal bone were detected in 8.2% and 20.4% of cases, respectively, by three-dimensional ultrasound, whereas all cases evaded detection with two-dimensional ultrasound (p < 0.001 and p = 0.001, respectively). CONCLUSIONS: Fetal nasal bone length measured with two-dimensional ultrasound does not differ significantly from three-dimensional measurements. However, three-dimensional ultrasound is superior in detecting unilateral nasal bone hypoplasia or absence and in assessing fetal nasal bone shape. Hence, fetal nasal bone examination in the second trimester should include three-dimensional ultrasound evaluation.

The distribution of TPX2 in dividing leaf cells of the fern Asplenium nidus.

Plant cell division requires the dynamic organisation of several microtubule arrays. The mechanisms of regulation of the above arrays are under rigorous research. Among several factors that are involved in plant microtubule dynamics, the Targeting Protein for Xklp2 (TPX2) has been found to play a role in spindle organisation, in combination with Aurora kinases, in dividing cells of angiosperms. Microtubule organisation in dividing cells of ferns exhibits certain peculiarities. Accordingly, the presence and distribution of a TPX2 homologue might be helpful in understanding the patterns and regulatory mechanisms of microtubule arrays in this plant group. In this study, a putative TPX2 homologue was identified using Western blotting in the fern Asplenium nidus. It was found, using immunostaining and CLSM, that it is co-localised with perinuclear preprophase microtubules and the prophase spindle, and follows the microtubule pattern during metaphase/anaphase and telophase. During cytokinesis, while in angiosperms TPX2 is degraded, in A. nidus the TPX2 signal persists, co-localising with the phragmoplast. In early post-cytokinetic cells, a TPX2 signal is present on the nuclear surface facing the daughter cell wall and, thereafter it is co-localised with the fern-specific microtubule aggregation that lines the new wall, which is possibly involved in cortical microtubule assembly.

Tungsten affects the cortical microtubules of Pisum sativum root cells: experiments on tungsten-molybdenum antagonism.

Tungsten (W) is increasingly shown to be toxic to various organisms, including plants. Apart from inactivation of molybdo-enzymes, other potential targets of W toxicity in plants, especially at the cellular level, have not yet been revealed. In the present study, the effect of W on the cortical microtubule array of interphase root tip cells was investigated, in combination with the possible antagonism of W for the pathway of molybdenum (Mo). Pisum sativum seedlings were treated with W, Mo or a combination of the two, and cortical microtubules were examined using tubulin immunofluorescnce and TEM. Treatments with anti-microtubule (oryzalin, colchicine and taxol) or anti-actomyosin (cytochalasin D, BDM or ML-7) drugs and W were also performed. W-affected cortical microtubules were low in number, short, not uniformly arranged and were resistant to anti-microtubule drugs. Cells pre-treated with oryzalin or colchicine and then treated with W displayed W-affected microtubules, while cortical microtubules pre-stabilized with taxol were resistant to W. Treatment with Mo and anti-actomyosin drugs prevented W from affecting cortical microtubules. Cortical microtubule recovery after W treatment was faster in Mo solution than in water. The results indicate that cortical microtubules of plant cells are indirectly affected by W, most probably through a mechanism depending on the in vivo antagonism of W for the Mo-binding site of Cnx1 protein.

Sinuous ordinary epidermal cells: behind several patterns of waviness, a common morphogenetic mechanism.

Morphogenesis of sinuous epidermal cells in leaves of the fern Asplenium nidus and the monocotyledonous Cyperus papyrus, petals of the dicotyledonous Begonia lucerna. and in-vitro-grown leaves of the fern Adantum capillus-veneris is controlled by the local differentiation of their walls. In all these cases wall pads, including radial cellulose microfibrils, arc deposited at the junctions of the external periclinal wall with the anticlinal ones. Moreover, in Asplenium nidus, similar wall pads form at the junctions of the internal periclinal wall with the anticlinal ones. The wall pads are connected to anticlinal cellulose microfibril bundles running the whole depth of the anticlinal walls nr part of it. This wall differentiation imposes a highly controlled cell wall expansion, a consequence of which is the waviness of the epidermal cell anticlinal, walls. The pattern of wall reinforcement varies among different species, resulting in differences in the pattern of waviness. Cortical microtubule arrays mirror the orientated deposition of cellulose microfibrils in the epidermal Cells. These findings, derived from plants from different major groups, show a common epidermal cell morphogenetic mechanism depending on radial cellulose microfibrils and cellulose microfibril bundles. The facts that (a) epidermal cell morphogenesis in Adiantum copillus-veneris leaves grown in vitro differs considerably from that of typical leaves and (b) petal epidermal cells in Begonia lucerna are sinuous, while leaf epidermal cells are not, suggest that this mechanism may he affected by epigenetic factors.

Microtubule organization and cell morphogenesis in two semi-lobed cell types of Adiantum capillus-veneris L. leaflets.

Protodermal cells of Adiantum capillus-veneris leaflets are polyhedral, displaying regularly arranged cortical microtubules transverse to the main cell axis. The nascent epidermal cells partly detach from the underlying mesophyll cells by formation of intercellular spaces, containing PAS-positive material. In early differentiating upper epidermal cells discrete U-like bundles of cortical microtubules form on the internal periclinal and the anticlinal walls. In contrast, microtubules are randomly scattered along the external periclinal wall. Microtubule bundles of neighbouring anticlinal walls of epidermal cells exhibit an alternating disposition but are directly opposite to those of underlying mesophyll cells. Epidermal cell wall is locally reinforced by thickenings arising under the microtubule bundles and including parallel cellulose microfibrils. The pattern of wall thickenings reflects that of the microtubule bundles. The internal periclinal epidermal cell region expands at the sectors free of wall thickenings, forming several lobes. Simultaneously, intercellular spaces open at the thickened regions of anticlinal walls, which finally become wavy. In contrast, the external periclinal wall does not form any lobes but remains smooth. As a result, epidermal cells become 'semi-lobed'. The lobes of lower epidermal cells are less prominent. Mesophyll cells surrounding the endodermis are also 'semi-lobed'. Their morphogenesis is achieved by the same mechanism. Colchicine treatment inhibits the 'semi-lobed' morphogenesis of epidermal cells and mesophyll cells surrounding the endodermis and the concomitant intercellular space opening. These observations reveal that the primary event of 'semi-lobed' cell morphogenesis is the organization of two different patterns of the cortical microtubule cytoskeleton in the same cell.