Jana Nebesářová

The Arabidopsis Exocyst Complex Is Involved in Cytokinesis and Cell Plate Maturation

The plant cell cytokinesis is driven from the onset by highly organized vesicle fusion resulting in cell plate and new cell wall formation separating daughter cells. The evolutionarily conserved exocyst complex regulating exocytic vesicle binding to the plasma membrane is involved in both the final separation of cells as in animals and also in the initiation of cell plate in plant cells. Cell reproduction is a complex process involving whole cell structures and machineries in space and time, resulting in regulated distribution of endomembranes, organelles, and genomes between daughter cells. Secretory pathways supported by the activity of the Golgi apparatus play a crucial role in cytokinesis in plants. From the onset of phragmoplast initiation to the maturation of the cell plate, delivery of secretory vesicles is necessary to sustain successful daughter cell separation. Tethering of secretory vesicles at the plasma membrane is mediated by the evolutionarily conserved octameric exocyst complex. Using proteomic and cytologic approaches, we show that EXO84b is a subunit of the plant exocyst. Arabidopsis thaliana mutants for EXO84b are severely dwarfed and have compromised leaf epidermal cell and guard cell division. During cytokinesis, green fluorescent protein–tagged exocyst subunits SEC6, SEC8, SEC15b, EXO70A1, and EXO84b exhibit distinctive localization maxima at cell plate initiation and cell plate maturation, stages with a high demand for vesicle fusion. Finally, we present data indicating a defect in cell plate assembly in the exo70A1 mutant. We conclude that the exocyst complex is involved in secretory processes during cytokinesis in Arabidopsis cells, notably in cell plate initiation, cell plate maturation, and formation of new primary cell wall.

Spermiogenesis in the pseudophyllid cestode Eubothrium crassum (Bloch, 1779).

Abstract. Spermiogenesis of the pseudophyllidean tapeworm Eubothrium crassum has been described by the aid of transmission electron microscopy for the first time. Initially, early spermatids form a distal cytoplasmic protrusion, a differentiation zone containing a small electron-dense, apically oriented region. Out of this region, two centrioles with rootlets develop. The centrioles become orientated in the same plane with the appearance of an intercentriolar body. Now, the long axes of the rootlets are parallel with each other and with the long axis of the nucleus. Two flagella of subsequently unequal length are formed very rapidly. Simultaneously, a median cytoplasmic process (MCP) develops distal to the flagella. Two arching membranes appear at the base of the differentiation zone. Each flagellum, still being in contact with an intercentriolar body, rotates to a position parallel with the MCP. The nucleus migrates very rapidly into the MCP at this stage. Subsequently, the two flagella fuse with the MCP. Finally, the basal bodies with the rootlets detach from the flagella, the intercentriolar body changes its structure and spermatids are pinched off from a condensing residual cytoplasm at the level of the arching membranes.

Spermiogenesis in the proteocephalidean cestode Proteocephalus torulosus (Batsch, 1786)

Spermiogenesis of the proteocephalidean cestode Proteocephalus torulosus (Batsch, 1786) was examined for the first time using transmission electron microscopy. Spermiogenesis begins with the formation of a distal cytoplasmic protrusion, a differentiation zone, at the periphery of the early spermatid. This differentiation zone is lined with cortical microtubules and contains two centrioles aligned along the same axis. Subsequently, each centriole is associated with the striated root and the intercentriolar body appears between them. A flagellar bud arises from each centriole, growing later as a free flagellum. Simultaneously, a median cytoplasmic process (MCP) develops distally to the flagella. The two flagella, which are of unequal length, become longer and rotate towards the MCP. At this stage, two arching membranes appear at the base of the differentiation zone. The nucleus elongates and when both flagella are fused with the MCP, the nucleus subsequently migrates into the MCP. Finally, the advanced spermatids detach from a condensing residual cytoplasm at the level of the arching membranes.

Association of Poly(4-hydroxystyrene)-block-Poly(Ethylene oxide) in Aqueous Solutions: Block Copolymer Nanoparticles with Intermixed Blocks

Association behavior of diblock copolymer poly(4-hydroxystyrene)-block-poly(ethylene oxide) (PHOS-PEO) in aqueous solutions and solutions in water/tetrahydrofuran mixtures was studied by static, dynamic, and electrophoretic light scattering, (1)H NMR spectroscopy, transmission electron microscopy, and cryogenic field-emission scanning electron microscopy. It was found that, in alkaline aqueous solutions, PHOS-PEO can form compact spherical nanoparticles whose size depends on the preparation protocol. Instead of a core/shell structure with segregated blocks, the PHOS-PEO nanoparticles have intermixed PHOS and PEO blocks due to hydrogen bond interaction between -OH groups of PHOS and oxygen atoms of PEO and are stabilized electrostatically by a fraction of ionized PHOS units on the surface.

Ultrastructure of the early rostellum of Silurotaenia siluri (Batsch, 1786) (Cestoda: Proteocephalidae)

The neodermis of the whole early rostellum of Silurotaenia siluri (Batsch, 1786) bears filamentous microtriches. At the base, there are five to six irregular rows of hooks and spine-like microtriches. The rostellar hooks of S. siluri originate through the enlargement of microtriches. Electron-dense hook substance is deposited along the edges of the microthrix to form the hook blade and basal plate. The blade does not become hollow as in the cyclophyllidean cestodes. The basal plate of the hook, corresponding to the handle and the guard of Cyclophyllidea, is formed by the deposition of hook substance around the base of the microthrix. Within the centre of the base, only a narrow cleft-like core is occupied by distal cytoplasm. The hook bases are more deeply inserted into the distal cytoplasm, as the bases of the spine-like microtriches are localized on the rostellum below the rows of hooks. No hemidesmosomes, fixing the hook base to the basal lamina, are present. Eccrine gland cells and uniciliate and nonciliate sensory receptors of the primitive rostellum are described.

Simultaneous detection of multiple targets for ultrastructural immunocytochemistry

Simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. While fluorescent microscopy allows for simultaneous detection of multiple antigens, the sensitive electron microscopy immunodetection is limited to only two antigens. In order to overcome this limitation, we prepared a set of novel, shape-coded metal nanoparticles readily discernible in transmission electron microscopy which can be conjugated to antibodies or other bioreactive molecules. With the use of novel nanoparticles, various combinations with commercial gold nanoparticles can be made to obtain a set for simultaneous labeling. For the first time in ultrastructural histochemistry, up to five molecular targets can be identified simultaneously. We demonstrate the usefulness of the method by mapping of the localization of nuclear lipid phosphatidylinositol-4,5-bisphosphate together with four other molecules crucial for genome function, which proves its suitability for a wide range of biomedical applications.

Early intrauterine embryonic development in Khawia sinensis Hsü, 1935 (Cestoda, Caryophyllidea, Lytocestidae), an invasive tapeworm of carp (Cyprinus carpio): an ultrastructural study

Intrauterine embryonic development in the caryophyllidean tapeworm Khawia sinensis has been investigated using transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate for glycogen. Contrary to previous light microscopy findings that reported the release of non-embryonated eggs of K. sinenesis to the external environment, the present study documents various stages of embryonation (ovoviviparity) within the intrauterine eggs of this cestode. At the initial stage of embryonic development, each fertilised oocyte is accompanied by several vitellocytes that become enclosed within the operculate, electrondense shell. Cleavage divisions result in formation of blastomeres (up to about 24 cells) of various sizes. Mitotic divisions and apparent rosette arrangment of the blastomeres, the latter atypical within the Eucestoda, are observed for the first time in the intrauterine eggs of K. sinenesis. The early embryo enclosed within the electrondense shell is surrounded by a thin membraneous layer which in some enlarged regions shows presence of nuclei. Simultaneously to multiplication and differentiation, some of the blastomeres undergo deterioration. A progressive degeneration of the vitellocytes within eggs provides nutritive reserves, including lipids, for the developing embryo. The possible significance of this atypical timing of the intrauterine embryonic development to (1) the ecology of K. sinensis and that of a recent introduction of another invasive tapeworm, the caryophyllidean Atractolytocestus huronensis Anthony, 1958 to Europe; and (2) the affiliation of caryophyllideans with other lower cestodes, are discussed.

Ultrastructure of the mature spermatozoon of Eubothrium rugosum (Batsch, 1786) with a re-assessment of the spermatozoon ultrastructure of Eubothrium crassum (Bloch, 1779) (Cestoda: Bothriocephalidea)

SummaryThe ultrastructure of the mature spermatozoon of the bothriocephalidean tapeworm Eubothrium rugosum, a parasite of the burbot, Lota lota (L.), was studied by transmission electron microscopy for the first time. In addition, spermatozoon ultrastructure of Eubothrium crassum has been re-assessed. New is the finding, that the mature spermatozoa of both species of the genus Eubothrium exhibit essentially the same general morphology. They are filiform cells tapering at both extremities, and they possess the two axonemes with 9+“1” pattern of Trepaxonemata, attachment zones, a nucleus, cortical microtubules (CMs), electron-dense granules, and a single crested body. Structural polymorphism of the CBs has been found within the two Eubothrium species for the first time. The anterior ring of electron-dense tubular structures surrounding a single axoneme marks the border between the two defined regions, region I and region II of the spermatozoon. This unique feature has only been observed in the Bothriocephalidea. The anuclear axoneme region II of Eubothrium spermatozoa fluently verges into a nuclear region III. The posterior part of the spermatozoon contains one-axoneme, few CMs and a posterior extremity of the nucleus that subsequently disappears. The posterior extremity of the male gametes of the genus Eubothrium exhibits elements of a disorganized axoneme which characterize also spermatozoa of the family Triaenophoridae. Discussed are interspecific similarities and differences between the spermatozoa of the two Eubothrium species as well as between these and other Eucestoda.

Cellular interfaces with hydrogen-bonded organic semiconductor hierarchical nanocrystals

Successful formation of electronic interfaces between living cells and semiconductors hinges on being able to obtain an extremely close and high surface-area contact, which preserves both cell viability and semiconductor performance. To accomplish this, we introduce organic semiconductor assemblies consisting of a hierarchical arrangement of nanocrystals. These are synthesised via a colloidal chemical route that transforms the nontoxic commercial pigment quinacridone into various biomimetic three-dimensional arrangements of nanocrystals. Through a tuning of parameters such as precursor concentration, ligands and additives, we obtain complex size and shape control at room temperature. We elaborate hedgehog-shaped crystals comprising nanoscale needles or daggers that form intimate interfaces with the cell membrane, minimising the cleft with single cells without apparent detriment to viability. Excitation of such interfaces with light leads to effective cellular photostimulation. We find reversible light-induced conductance changes in ion-selective or temperature-gated channels.Nanomaterials that form a bioelectronic interface with cells are fascinating tools for controlling cellular behavior. Here, the authors photostimulate single cells with spiky assemblies of semiconducting quinacridone nanocrystals, whose nanoscale needles maximize electronic contact with the cells.

Size determination of Acipenser ruthenus spermatozoa in different types of electron microscopy.

In this study three types of scanning electron microscopes were used for the size determination of spermatozoa of sterlet Acipenser ruthenus - high vacuum scanning electron microscope (SEM, JEOL 6300), environmental scanning electron microscope (ESEM, Quanta 200 FEG), field emission scanning electron microscope (FESEM, JEOL 7401F) with cryoattachment Alto 2500 (Gatan) and transmission electron microscope (TEM, JEOL 1010). The use of particular microscopes was tied with different specimen preparation techniques. The aim of this study was to evaluate to what degree the type of used electron microscope can influence the size of different parts of spermatozoa. For high vacuum SEM the specimen was prepared using two slightly different procedures. After chemical fixation with 2.5% glutaraldehyde in 0.1M phosphate buffer and post-fixation by 1% osmium tetroxide, the specimen was dehydrated by acetone series and dried either by critical point method or by means of t-butylalcohol. For ESEM fresh, unfixed material was used, which was dropped on microscopic copper grids. In FESEM working in cryo-mode the specimen was observed in a frozen state. Ultrathin sections from chemically fixed and Epon embedded specimens were prepared for TEM observation. Distinct parts of sterlet spermatozoa were measured in each microscope and the data obtained was statistically processed. Results confirmed that the classical chemical procedure of specimen preparation for SEM including critical point drying method led to a significant contraction of all measured values, which could deviate up to 30% in comparison with values measured on the fresh chemically untreated specimen in ESEM. Surprisingly sperm dimensions determinated on ultrathin sections by TEM are comparable with values obtained in ESEM or FESEM.