Immunocytochemical localization of lysozyme in the nephrocytes of the harvestman, Leiobunum rotundum

Nephrocytes are cells involved in the metabolism of hemolymph components and are characterized by peripheral finger-like projections bordering a labyrinthine channel system. The antibacterial protein lysozyme was localized in anterior and pericardial nephrocytes of the harvestman, Leiobunum rotundum, by biochemical and immuno-gold postembedding-labelling techniques. Lysozyme-activity was demonstrated in experimental animals, challenged by Micrococcus luteus Grampositive bacteria. With SDS-electrophoresis, lysozyme was proved to be present in nephrocytes adjoining the heart, before studies at the electron microscopical level were carried out. The opilionid lysozyme was found to have a molecular weight of 14000 Da, thus belonging to the c-type lysozyme which is represented by hen-egg-white lysozyme. In untreated animals the presence of lysozyme could not be proved by electrophoresis. With immunogold labelling, however, lysozyme could be clearly localized in the vesicles of nephrocytes of untreated animals. Additional control experiments were carried out which revealed that the nephrocytes of L. rotundum in vivo are able to take up native lysozyme from an exterior medium. Therefore, the problem needs to be solved if nephrocyte vesicles transport lysozyme into or out of the cell.     

The ultrastructure and possible functions of nephrocytes in the trombiculid mite Hirsutiella zachvatkini (Acariformes: Trombiculidae)

The ultrastructure of nephrocytes during the post-larval development of the trombiculid mite Hirsutiella zachvatkini (Schluger) was investigated by means of transmission electron microscopy. Nephrocytes are situated either individually or in groups inside the haemocoelic space and are always ensheathed by a basal membrane. They contain numerous tubular elements, electron-dense inclusions and sometimes electron-lucent vacuoles. Invaginations of the plasma membrane, often in the form of labyrinthine channels, with coated pits of plasma membrane and characteristic slit diaphragms linking adjacent pedicels, typically occupy the peripheral parts of the cells. However, no distinct zonation of organelles in the nephrocytes of trombiculids was observed. The number of nephrocytes in the haemocoel and the intensity of development of their tubular elements and in particular of labyrinthine channels vary significantly during the ontogenesis of mites. The possible functions of nephrocytes and their structure and differentiation in comparison with other arachnids are discussed.     

The fine structure of the kidney of Achatina achatina (L.)

Summary The kidney sac of Achatina achatina, the site of primary urine formation, seems to contain no direct structural analogue of the vertebrate glomerular podocytes. The nephrocytes which line the kidney sac and separate the blood from the primary urine are supported by a basal lamina which is permeable to ferritin but impermeable to colloidal gold particles (ca. 100 Å, and 80–240 Å respectively). The blood capillaries within the kidney sac are of two types, fenestrated and unfenestrated. The basal lamina which surrounds them is impermeable to haemocyanin. The nephrocytes are then, bathed apically by primary urine and basally by an ultrafiltrate of the blood. It is proposed that fluid enters the urinary space from the connective tissue by passing between the nephrocytes, perhaps through pores in the septate junctions. Other possible mechanisms of primary urine formation are discussed. The nephrocytes contain peroxisomes which may be involved in urate metabolism.The cells of the ureteral epithelium bear a lumenal microvillous border. Their lateral and basal plasma membranes are elaborately folded. These cytoplasmic folds enclose extracellular channels through which fluid is transported from the urine back into the blood.     

Organization of a phyllobranchiate gill from the green shore crab Carcinus maenas (Crustacea,Decapoda)

Summary The phyllobranchiate gills of the green shore crab Carcinus maenas have been examined histologically and ultrastructurally. Each gill lamella is bounded by a chitinous cuticle. The apical surface of the branchial epithelium contacts this cuticle, and a basal lamina segregates the epithelium from an intralamellar hemocoel. In animals acclimated to normal sea water, five epithelial cell types can be identified in the lamellae of the posterior gills: chief cells, striated cells, pillar cells, nephrocytes, and glycocytes. Chief cells are the predominant cells in the branchial epithelium. They are squamous or low cuboidal and likely play a role in respiration. Striated cells, which are probably involved in ionoregulation, are also squamous or low cuboidal. Basal folds of the striated cells contain mitochondria and interdigitate with the bodies and processes of adjacent cells. Pillar cells span the hemocoel to link the proximal and distal sides of a lamella. Nephrocytes are large, spherical cells with voluminous vacuoles. They are rimmed by foot processes or pedicels and frequently associate with the pillar cells. Glycocytes are pleomorphic cells packed with glycogen granules and multigranular rosettes. The glycocytes often mingle with the nephrocytes. Inclusion of the nephrocytes and glycocytes as members of the branchial epithelium is justified by their participation in intercellular junctions and their position internal to the epithelial basal lamina.     

Probable role of dopamine in the regulation of ventral nephrocyte functioning in Drosophila melanogaster adults

Filtration of haemolymph in insects to remove waste products is performed by nephrocytes, which comprise accessory cells of the circulatory system that are not connected to Malpighian tubules. There are two types of nephrocytes in Drosophila: ventral cells, situated around the junction between the cardia and the oesophagus, and pericardial cells, situated around the heart. In the present study, the expression of dopamine D1 (DopR) and D2‐like (DD2R) receptors in the ventral nephrocytes of Drosophila melanogaster Meigen (Diptera: Drosophilidae) is investigated. Immunohistochemical staining with polyclonal antibodies against DopR and DD2R demonstrates the presence of these receptors in adult nephrocytes. The functioning of D. melanogaster nephrocytes is investigated by evaluation of mortality rates in flies treated with silver nitrate (AgNO₃) compared with untreated controls. To determine whether a change in the level of dopamine receptors has an effect on the functioning of nephrocytes, the antisense suppressor of DD2R gene together with a nephrocyte‐specific driver is used in the UAS‐GAL4 system. The suppression of DD2R in nephrocytes results in a significant decrease of mortality under toxic conditions. Taken together, the data obtained in the present study indicate that dopamine takes part in the control of ventral nephrocyte functioning in D. melanogaster.     

Spatio-temporal reorganization of intracytoplasmic microtubules is associated with nuclear selection and differentiation during the developmental process in the ciliateTetrahymena thermophila

Summary Indirect immunofluorescence has revealed various intracytoplasmic microtubular structures, which are transiently polymerized in specific subcellular locations during the developmental process of conjugation in the ciliateTetrahymena thermophila. These structures include: (1) micronuclear spindles, (2) perimicronuclear microtubules, (3) microtubular baskets surrounding migrating pronuclei, and (4) microtubules interconnecting the pronuclei with the conjugants' junctional zone. Furthermore, a peripheral network of intracytoplasmic microtubules related to the cell cortex is present in both vegetative cells and in conjugants. Comparative observations made on cells undergoing normal conjugation and defective conjugation (occurring either spontaneously or induced by taxol) has revealed some rules governing the pattern of deployment of conjugation-specific microtubules. The presence of perinuclear microtubular arrays during early postmeiotic stages of development is strictly limited to more anteriorly located nuclei which includes the selected haploid nucleus that further divides to form the stationary and migratory pronuclei. These perinuclear microtubules may be involved in the positional control of nuclear fates leading to effective nuclear selection. Microtubular bundles associated with pronuclei and connecting the junctional zone are only formed in the presence of functional pronuclei, and may be involved in the guidance of pronuclei leading to their fusion. The mechanism of cytoplasmic control of nuclear differentiation of derivatives of the zygotic nucleus appear to be associated with a coordinate action of two microtubular arrays: spindle microtubules of the second postzygotic division and the peripheral intracytoplasmic network of microtubules, leading to a proper subcortical positioning of the postzygotic nuclei at opposite poles of the cell.Abbreviations MTs Microtubules     

Involvement of the cytoskeleton in the intracellular distribution of mannoproteins in hyphal cells ofCandida albicans

Cylindrical growth of fungal hyphae requires spatial organization of secretion to the growing tip. In order to better understand the involvement of the cytoskeleton in the spatial control of the secretion, we examined the effects of two anti-cytoskeletal drugs, benomyl and cytochalasin A, on the intracellular distribution of mannoproteins, a major secreted component of the cell wall, in hyphal cells of the dimorphic yeastCandida albicans. The distribution of the mannoproteins was assessed by epifluorescence microscopy with a fluorescence-labelled lentil lectin (FITC-LCA). Brefeldin A, an inhibitor of secretory transport, induced a localized accumulation of the mannopolysaccharides near the tip as previously reported (Akashiet al. 1997). Benomyl, an inhibitor of microtubules, disrupted the localized accumulation of the polysaccharides. Cytochalasin A, an inhibitor of actin, caused a localized accumulation of the polysaccharides near the tip, where Golgi-like cisternae were also accumulated. Both cytochalasin A and brefeldin A caused some modifications of the actinnnetwork, but neither disturbed the polarization of actin and neither affected the microtubule network. Our results suggested that the microtubules are involved in membrane trafficking in hyphal growth as well as the cell polarity of the hyphae.     

Microtubules do not control orientation of secondary cell wall microfibril deposition inMicrasterias

Summary The influence of the microtubule disorganizing substances amiprophos-methyl (APM) and colchicine on secondary wall formation inMicrasterias denticulata was investigated by the freezeetch technique. The results reveal that neither microtubule inhibitor changes the pattern of microfibril deposition. The application of APM or colchicine also does not cause any structural alterations of the microfibrils or of the protoplasmic (Pf) and the exoplasmic (Ef) fracture face of the plasma membrane, thus indicating that microtubules are not involved in secondary wall formation inM. denticulata. However, since areas of the plasma membrane which collapsed upon freeze-etching are restricted to the Pf-face of cells treated with microtubule inhibitors, cortical microtubules may function as mechanical support during secondary wall formation. In the cortical cytoplasm filamentous structures are found in close spatial relationship and an almost parallel alignment to rosettes of the plasma membrane.     

Microtubule reorganization,cell wall synthesis and establishment of the axis of elongation in regenerating protoplasts of the algaMougeotia

Summary Microtubule reorganization and cell wall deposition have been monitored during the first 30 hours of regeneration of protoplasts of the filamentous green algaMougeotia, using immunofluorescence microscopy to detect microtubules, and the cell-wall stain Tinopal LPW to detect the orientation of cell wall microfibrils. In the cylindrical cells of the alga, cortical microtubules lie in an ordered array, transverse to the long axis of the cells. In newly formed protoplasts, cortical microtubules exhibit some localized order, but within 1 hour microtubules become disordered. However, within 3 to 4 hours, microtubules are reorganized into a highly ordered, symmetrical array centered on two cortical foci. Cell wall synthesis is first detected during early microtubule reorganization. Oriented cell wall microfibrils, co-aligned with the microtubule array, appear subsequent to microtubule reorganization but before cell elongation begins. Most cells elongate in the period between 20 to 30 hours. Elongation is preceded by the aggregation of microtubules into a band intersecting both foci, and transverse to the incipient axis of elongation. The foci subsequently disappear, the microtubule band widens, and microfibrils are deposited in a band which is co-aligned with the band of microtubules. It is proposed that this band of microfibrils restricts lateral expansion of the cells and promotes elongation. Throughout the entire regeneration process inMougeotia, changes in microtubule organization precede and are paralleled by changes in cell wall organization. Protoplast regeneration inMougeotia is therefore a highly ordered process in which the orientation of the rapidly reorganized array of cortical microtubules establishes the future axis of elongation.     

Tracheid differentiation in tobacco pith cultures

Summary Sterile pith cultures of Nicotiana tabacum have been induced to form localized regions of differentiating tracheids. These localized regions have been examined by phase, fluorescence, and electron microscopy, and polarization optics. Fixation for electron microscopy was with glutaraldehyde-osmium. The differentiating tracheids develop characteristic thick cell walls which are eventually lignified. The lignifications appear to be uniform throughout the secondary wall and little or no lignin appears to be deposited in the primary walls or intercellular layer. At all stages of secondary wall deposition, the peripheral cytoplasm contains a system of microtubules which form a pattern similar to that of the developing thickenings. Within this system the microtubules are oriented, the direction of orientation mirroring that of the fibrils in the most recently deposited parts of the wall. The observations support the view that the microtubules are somehow involved in microfibril orientation. The microtubules appear to be attached to the plasma membrane which has a triple layered structure. The two electron dense layers of the plasma membrane have a particulate structure. In the differentiating tracheids at regions where secondary wall thickening has not yet been deposited numerous invaginations of the plasma membrane are observed which contain loosely organized fibrillar material. It is suggested that these are areas of localized activity of the plasma membrane and that the enzymes concerned with the final organization of the cellulose microfibrils are situated at the surface of the plasma membrane. Dictyosomes in the differentiation cells give rise to vesicles which contain fibrous material and the contents are incorporated into the cell wall. Numerous profiles characteristic of plasmodesmata are evident in sections of the secondary thickenings.Part of this work was carried out at the Osborne Memorial Laboratories, Yale University.     

The convergence of Notch and MAPK signaling specifies the blood progenitor fate in the Drosophila mesoderm

Blood progenitors arise from a pool of pluripotential cells (“hemangioblasts”) within the Drosophila embryonic mesoderm. The fact that the cardiogenic mesoderm consists of only a small number of highly stereotypically patterned cells that can be queried individually regarding their gene expression in normal and mutant embryos is one of the significant advantages that Drosophila offers to dissect the mechanism specifying the fate of these cells. We show in this paper that the expression of the Notch ligand Delta (Dl) reveals segmentally reiterated mesodermal clusters (“cardiogenic clusters”) that constitute the cardiogenic mesoderm. These clusters give rise to cardioblasts, blood progenitors and nephrocytes. Cardioblasts emerging from the cardiogenic clusters accumulate high levels of Dl, which is required to prevent more cells from adopting the cardioblast fate. In embryos lacking Dl function, all cells of the cardiogenic clusters become cardioblasts, and blood progenitors are lacking. Concomitant activation of the Mitogen Activated Protein Kinase (MAPK) pathway by Epidermal Growth Factor Receptor (EGFR) and Fibroblast Growth Factor Receptor (FGFR) is required for the specification and maintenance of the cardiogenic mesoderm; in addition, the spatially restricted localization of some of the FGFR ligands may be instrumental in controlling the spatial restriction of the Dl ligand to presumptive cardioblasts.     

Microtubule organization differs between acid and alkaline bands in internodal cells ofChara but bands can develop in the absence of microtubules

We have studied the relationship between pH banding and the organization of cortical microtubules in the alga Chara corallina Klein ex Willd. Microtubules were visualized by immunofluorescence and also by imunogold-silver enhancement to allow immediate comparison of microtubule arrangement with visible structural cell features. In cells that are nearing growth completion, microtubule number and alignment change between acidic and alkaline bands over a distance of a few micrometres. Thus, it appears that the still unknown mechanisms for microtubule organization respond to the localized differences in membrane properties. Band formation was not prevented when microtubules were depolymerized with the herbicide oryzalin, demonstrating that microtubules are not necessary for pH bands to develop in these cells.Abbreviations DMSO dimethylsulfoxide - MT microtubule We thank Frank Gubler for helpful advice on immunogold-silver enhancement procedures, Brian Gunning for tuition in confocal microscopy, Ann Cork for assistance with photography and Dean Price for helpful discussions. G.O.W. gratefully acknowledges the receipt of a National Research Fellowship and a Queen Elizabeth II Fellowship from the Australian Research Council.     

Cytoskeleton,cell surface and the development of invasive plasmodial tapetum inTradescantia virginiana L.

Summary The anther tapetum inTradescantia virginiana L. is of the invasive plasmodial type: the cells lose their walls during early spore meiosis and develop long invasion processes which invade the loculus to penetrate spaces between the sporogenous cells. Fusion to form a syncytium is delayed and conventional ultra-thin sections and the Thiéry reaction reveal the presence of a loose fibrillar extracellular cell coat on the free surfaces of tapetal cells and their invasion processes. Cell fusion involves formation of apposition areas characterized by an absence of cell coat and the local appearance of microtubular arrays. Conspicuous membrane sacs, associated closely with microtubules, were found to migrate to and accumulate at the plasma membranes near the fusion sites and sporogenous cells. Microtubules are always present in the cortical regions of the tapetal cells and their invasion processes. It is surmised that microtubules are not responsible either for initiating or guiding tapetal invasion of the loculus; instead they may help to sustain the form of the invasion processes, help in the migration of membrane sacs, and participate in cell fusion. The cell coat disappears with syncytium formation towards the end of meiosis, and the developing spore cells become surrounded by a perispore membrane, which, derived from the original plasma membranes and augmented by membrane sacs, forms labyrinthine membrane reservoirs that are described further in the accompanying paper.     

Where and when is microtubule diversity generated inParamecium? Immunological properties of microtubular networks in the interphase and dividing cells

Summary Ciliates are highly differentiated cells which display extensive deployment of microtubular systems. Because genetic diversity of tubulin is extremely reduced in these cells, microtubule diversity is mostly generated at the post-translational level either through direct modification of tubulin or through the binding of associated proteins to microtubules. We have undertaken a systematic exploration of microtubule diversity in ciliates by way of production of monoclonal antibodies. Previously we reported the biochemical characterization of these antibodies. In addition to antibodies directed against primary sequences, we obtained antibodies directed against post-translational modifications. In this paper, we report a detailed analysis of the distribution of the various epitopes on the microtubular networks ofParamecium, both in interphase cells and during division morphogenesis. Each of these antibodies decorates a subset of microtubules. Acetylation, recognized by antibodies TEU 318 and TEU 348, is detected on stable microtubules early after microtubule assembly. Epitopes recognized by two other antibodies (TAP 952 and AXO 58) are found on a subset of stable microtubules; in addition, the TAP 952 antibody is also found on labile microtubules; both epitopes are detected as soon as microtubule assembly occurs. In contrast, the epitope of the antibody, AXO 49, is associated with only a restricted subset of stable microtubules in the interphase cell, and is detected a lag-time after microtubule assembly during division morphogenesis. These data show that microtubule diversity is generated through a time-dependent sequence and according to a definite spatial pattern.     

Structure and function of the microtubular cytoskeleton during pollen development in Gasteria verrucosa (Mill.) H. Duval

In a study of pollen development in Gasteria verrucosa, the changes in the spatial organization of microtubules were related to the processes of cell division, nuclear movement and cytomorphogenesis. Sections of polyethylene-glycol-embedded anthers of G. verrucosa were processed immunocytochemically to record the structure and succession of fluorescently labeled microtubular configurations. Using microspectrophotometric measurements the relative quantity of tubulin in microtubules per unit of cytoplasm was determined. Cell dimensions and nuclear positions were measured to relate changes in cell shape and nuclear movements to microtubular configurations. Microtubules were detected in the different cells during microsporogenesis and microgametogenesis. In microspore mother cells which are approximately isodiametric at interphase, microtubules were predominantly arranged in a criss-cross pattern. The microtubules probably function as a flexible cytoskeleton which sustains the integrity of the cytoplasm. Bundles of microtubules were observed in the microspores, in the generative cells and during nuclear division, where they functioned in establishing and maintaining cell and spindle shapes. Microtubules radiating from nuclear membranes appeared to fix the nucleus in position. In prophase of meiosis and after microspore mitosis, periods a high fluorescence intensity were distinguished indicating a variation in the quantity of microtubules.Abbreviation MT microtubule     

The Peripheral Vesicles of Trophozoites of the Primitive ProtozoanGiardia lambliaMay Correspond to Early and Late Endosomes and to Lysosomes

Giardia lamblia,a primitive eukaryotic cell, lacks organelles such as mitochondria, peroxisomes, and a typical Golgi complex and presents a system of vesicles located below the plasma membrane. We used fluorescence and electron microscopy to better characterize the peripheral vesicles. Incubation of living cells with acridine orange showed that the peripheral vesicles correspond to an acidic compartment. Incubation with lucifer yellow, and with horseradish peroxidase, showed labeling of the peripheral vesicles even after several hours. Acid phosphatase was localized in the endoplasmic reticulum and in most of the peripheral vesicles. On the other hand, glucose 6-phosphatase, an endoplasmic reticulum marker, was observed in the endoplasmic reticulum cisternae and in some peripheral vesicles. A similar labeling pattern was observed using the zinc iodide technique, which reveals SH-containing proteins. Three-dimensional reconstruction and electron microscopy tomography of cells stained for acid phosphatase and glucose-6-phosphatase revealed the connection between some vesicles and profiles of the endoplasmic reticulum. Taken together, our observations suggest that trophozoites ofG. lambliapresent an endosomal–lysosomal system concentrated in a single system, the peripheral vesicles, which may represent an ancient organellar system that later on subdivided into compartments such as early and late endosomes and lysosomes.     

Spatial organization of microtubules in various types of cells in the embryonic tectal plate of mouse using immunofluorescence after PEG embedding

The spatial organization of microtubules in mitotic as well as in interphase cells and in axons has been investigated in situ in the embryonic nervous system of mice using high molecular weight polyethylene glycol-embedded semithin sections and immunofluorescence with a tubulin-specific polyclonal antibody. In situ, the overall process of mitosis appears nearly identical to that described in cell culture. All types of mitotic microtubules (kinetochore, interpolar and asterial) can be visualized at the different stages. The slight differences from observations in cell culture are explained by differences in cell interactions. In bipolar neuroepithelial cells, interphasic microtubules appear in the form of a framework surrounding the nucleus during its to-and-fro movements and which follows the modifications in shape of the cell processes. These microtubules seem to play an active role in the mechanism, indicating the modifications in length of the apical process. In the differentiating young neuron, tubulin increases in amount to be involved in the elongation of axonal microtubules. This increase seems to be independent of the presence of axons in the environment. Axonal microtubules are independent of a microtubule-organizing center localized in the perikaryon.     

Location,morphology and function of nephrocytes in termites

Insect nephrocytes are cells bathed in hemolymph and considered to have an excretory function. These cells have ambiguous nomenclature and are understudied in termites. This study is the first report on the occurrence, morphology and function of nephrocytes in different termite castes. Cytological characteristics in specific developmental stages and castes enable physiological functions to be inferred. Perforate diaphragms indicate a role in filtration, while the extensive peripheral invaginations of the cell membrane suggest active endocytosis. A sequence of morphologies in putative digestive vacuoles infers a lysosomal system and the occurrence of phosphatases suggests a function involving detoxification of substances sequestered from hemolymph. Pericardical nephrocytes took up the dye trypan blue injected in live termites, suggesting their activity connected to the filtration of the hemolymph. Additionally, histochemical tests showed the existence of stored proteins in their cytoplasm. These cells present a well-developed Golgi apparatus and abundant rough endoplasmic reticulum, consistent with protein synthesis. This study highlights the importance of nephrocytes in Isoptera and opens perspectives for further research of these cells.     

Evidence for initiation of microtubules in discrete regions of the cell cortex in Azolla root-tip cells,and an hypothesis on the development of cortical arrays of microtubules

Complexes of microtubules, vesicles, and (to varying degrees) dense matrix material around the microtubules were seen along the edges of cells in root apices of Azolla pinnata R.Br. (viewing the cells as polyhedra with faces, vertices and edges). They are best developed after cytokinesis has been completed, when the daughter cells are reinstating their interphase arrays of microtubules. They are not confined to edges made by the junction of new cell plates with parental walls, but occur also along older edges. Similar matrices and vesicles are seen amongst phragmoplast microtubules and where pre-prophase bands intersect the edges of cells. It is suggested that the complexes participate in the development of cortical arrays of microtubules. The observations are combined with others, made on pre-prophase bands and on the substructure of cortical arrays lying against the faces of cells, to develop an hypothesis on the development of cortical microtubules, summarised below: Microtubules are nucleated along the edges of cells, at first growing in unspecified orientations and then becoming bridged to the plasma membrane. Parallelism of microtubules in the arrays arises by inter-tubule cross-bridging. Lengths of microtubule are released from, or break off, the nucleating centres and are moved out onto the face of the cell by intertubule and tubule-membrane sliding, thus accounting for the presence there of short tubules with randomly placed terminations. The nucleating zones along cell edges might have vectorial properties, and thus be able to control the orientation of the microtubules on the different faces of the cell. Also, localised activation could generate localised arrays, especially pre-prophase bands in specified sites and planes. Two possible reasons for the spatial restriction of nucleation to cell edges are considered. One is that the geometry of an edge is itself important; the other is that along most cell edges there is a persistent specialised zone, inherited at cytokinesis by the daughter cells when the cell plate bisects the former pre-prophase-band zone.