Microtubular determination of cell shape during colony formation by the algaPediastrum

Summary Microtubules, which underlie the plasmalemma of zoospores ofPediastrum at the time of their aggregation to form daughter colonies, determine the shape of individual zoospores, and, as a consequence play a vital role in the patterned arrangement of cells in the colonies. Microtubules are also involved in extension of the characteristic horns on peripheral cells of colonies of this alga. Two densely staining plaques from which microtubules arise, are appressed to the plasmalemma at sites where horns emanate following adhesion of the zoospores. These plaques, which remain at the tips of the developing horns, are apparently microtubule organizing centres (MTOCs). IPC, a drug thought to affect MTOCs, interferes with the normal development of horns and the zoospores fail to aggregate in ordered arrays.     

Ultrastructural changes during zoospore formation in phytophthora parasitica

Cleavage of the multinucleate sporangial cytoplasm begins by a rearrangement and subsequent fusion of randomly dispersed cleavage vesicles. The vesicles line up in planes equidistant from neighboring nuclei and along the sporangial wall. In addition, they contribute to the enlargement of the central vacuole. Fusion of these vesicles with themselves and with the central vacuole cleaves the cytoplasm into uninucleate zoospores, each with two flagella. The sporangial wall consists of two layers, an outer thin one which is continuous over the plug of the discharge pore and an inner thick one which tapers off near the plug. The plug consists of fibrillar material and is ejected upon release of the zoospores. A plug-like structure separating the forming sporangium from the hypha has a homogeneous matrix pervaded with an anastomosing network of fine electron-dense channels. In addition, glycogen-like granules occur within mitochondria and paired structures are interpreted as procentrioles.     

Suberized tyloses in trees: An ultrastructural and cytochemical study

The nature of the wall layers observed in suberized tyloses was studied in Populus basalmifera L., Ulmus americana L. and Quercus rubra L. As the suberin layers were present only in tyloses that had completed their expansion, most of the results concern mature tyloses. The cyto- and immunocytochemical tests were conducted, respectively, with an exoglucanase having a binding affinity for β(1→4)-D-glucans, the subunits of cellulose, and with two monoclonal antibodies specific for un-esterified and esterified pectic molecules. In the three species, labelling for pectic compounds was intense over the external layer of tyloses but usually more dispersed or nearly absent over the layer corresponding to a primary wall that was, however, intensely labelled for β(1→4)-D-glucans. The outer wall layer, comparable to a middle lamella in mature tyloses, was continuous with similar material that appeared to be secreted by the tylosis. This material was particularly abundant in pit chambers, in void spaces between the tylosis and the vessel wall, particularly at the junction of the vessel and two adjacent cells, and close to the rim of vessel perforation plates. In P. balsamifera, a single suberized layer or occasionally a succession of suberized and cellulose-containing layers was observed internal to the tylosis primary wall. In U. americana, the wall of tylosis was similar to that of P. balsamifera except that, at times, a secondary-wall-like layer was formed and only a single suberized layer was observed. In Q. rubra, the suberized layer was always observed internal to the tylosis secondary wall. Simple pits were also constantly noted in Q. rubra tyloses. The occasional occurrence of a cellulosic layer internally to the suberized layer was observed in the three species. Histochemical tests revealed that lignin was also an important component of the tylosis wall. The tyloses frequently contained phenolic compounds in close association with the suberized layers. The significance of the formation of suberized tyloses in trees is discussed.     

Ultrastructural changes during sporangium formation and zoospore differentiation in Blastocladiella Emersonii

Samples from synchronized cultures of Blastocladiella emersonii were examined by electron microscopy from the late log phase to the completion of zoospore differentiation. Log-phase plants contain the usual cytoplasmic organelles but also have an unusual system of large tubules ca. 45 mμ diam that ramify in organized bundles throughout the protoplast. After induction, zoosporangium differentiation requires a 2-hr period in which the nuclei divide, a cross wall forms to separate the basal rhizoid region, and an apical papilla is produced. Nuclear division in B. emersonii is intranuclear with a typical microtubular spindle apparatus and paired, unequal, extranuclear centrioles at each pole. The papilla is formed by a process of localized cell wall breakdown and deposition of the papilla material by secretory granules. Differentiation of zoospores begins when one of the two centrioles associated with each nucleus elongates to form a basal body. The flagella fibers arise from the basal body and elongate into an expanding vesicle formed by the fusion of small secondary vesicles. The cleavage planes are formed by fusion of vesicles similar to those associated with flagellum initiation. When cleavage is complete, each sporangium contains ca. 250–260 uninucleate spore units with their flagella lying in the cleavage planes. Probable fusion of mitochondria to produce the single mitochondrion of the zoospore occurs after cleavage; the mitochondrion does not take its position around the basal body and rootlets until just before zoospore release. The ribosomal nuclear cap is organized and enclosed by a membrane formed through fusion of many small vesicles during a short period near the end of differentiation.     

Regional ultrastructural and cytochemical comparisons of the epithelial-mesenchymal interface during rat incisor development

A major theme in understanding epithelial-mesenchymal interactions during development focuses upon regional mesenchyme specification of epithelial differentiation. One particularly useful epidermal organ system for studying this issue is the rodent continuously growing and erupting incisor tooth organ. One advantage of this particular system resides in the regional features of the rodent incisor tooth organ. Along the labial surface, inner dental epithelial cells differentiate into ameloblasts that produce enamel extracellular matrix, whereas the epithelia along the lingual surface do not become ameloblasts and do not produce enamel matrix. This study has been designed to compare ultrastructural features of labial versus lingual surfaces, with particular emphasis upon mesenchymal cell shape, the orientation of extracellular matrix collagen, the basal lamina, and the distribution of sulfated glycoconjugates. Critical analyses of the data indicated that different microenvironments exist between epithelia and mesenchyme in the labial versus the lingual surfaces of the developing rodent incisor tooth organ.     

An ultrastructural and cytochemical analysis of oogenesis in the squid, Loligo pealei

Oogenesis has been investigated utilizing both light and electron microscopical techniques in the squid, Loligo pealei. This complex process has been divided into five stages according to the structure of the follicle. Because of the highly coordinated differentiation of the follicle cells (and follicular syncytium) and the oocyte, their development is described in concert. Specific attention is given to the contribution of the follicular syncytium to vitellogenesis and the formation of the extracellular egg envelope or chorion. Our observations indicate heterosynthetic yolk production and the synthesis of the secondary envelope by the follicular syncytium.     

An ultrastructural and cytochemical study of the wall-membrane apparatus of transfer cells using freeze-substitution

Summary A freeze-substitution technique is described which enables the ultrastructure of certain types of plant transfer cells to be preserved with minimal ice crystal damage. The ultrastructure of transfer cells fromFunaria, Lonicera, andSenecio after freeze-substitution has been compared with that of glutaraldehyde-osmium fixed material. The irregular clear zone between wall and plasma membrane, present in conventional preparations, is absent in freeze-substituted tissue. It is proposed that this interfacial zone is an artefact caused by expansion of wall ingrowth material during conventional fixation procedures. In transfer cells with a complex wall labyrinth the swelling of wall material severely disrupts the true structure of the wall-membrane apparatus and results in a large decrease in the surface to volume ratio of the protoplast. These findings are supported in the case ofFunaria by a freezefracture study. The reactivity of the plasma-membrane to the PTA/chromic acid stain is enhanced in freeze-substituted material. Use of theThiéry silver proteinate reagent in conjunction with freeze-substitution has revealed marked differences between the wall ingrowths ofFunaria sporophyte haustorium transfer cells and those ofLonicera nectary trichomes.     

Argyrophilic proteins in Dinoflagellates: An ultrastructural,cytochemical and immunocytochemical study

Summary— Dinoflagellate protists constitute an original eukaryotic phylum and have an ancestor in common with ciliates. They are important tools in studies of structure and function of the nucleus because they present a mixing of prokaryotic characteristics such as chromatin devoid of histones and nucleosomes, eukaryotic characteristics such as the presence of a nuclear membrane, nucleoli and AgNOR-like proteins and original characteristics of their own. Among them are the permanent compaction of the chromosomes, the presence of a nuclear envelope during the whole cell cycle, rare bases in their DNA, as well as an original mitosis. We have studied the distribution of the nuclear argyrophilic proteins (AgP) in three genera of Dinoflagellates (Prorocentrum, Crypthecodinium and Amphidinium) by means of light microscopy (LM) and electron microscopy (EM), using cytochemical silver staining and immunocytochemical reactions following various preparation procedures. By means of the silver staining reaction, we determined by LM the distribution of nucleoli in the three non-synchronized cell populations and localized by EM the presence of AgP. These are always found in the nucleolar fibrillo-granular compartment (FG) and partly in the chromosomes and in the nucleolar UCh (unwound region of the nucleolar chromosome corresponding to the NOR); the chromosomes and the UCh are always stained in P micans, under special conditions in C cohnii but never in A carterae. To determine whether these nucleolar and chromosomal proteins are similar or different, we modified the conditions of the silver staining reaction by acidic, alkaline or enzymatic pretreatments and changes in the reaction's temperature. Our results suggested that these proteins belong to different groups. We have characterized one of these proteins using a mammalian anti-B23 Ab in P micans cells. Positive labeling was mostly detected in chromosomes and UCh and in a smaller amount in the nucleolar FG and G compartments, co-locating with end-products of the silver staining reaction. This suggests that: i) one among the dinoflagellate chromosomal AgP is analogous to the B23 mammalian protein; and ii) this B23-like protein is probably a DNA partner.     

Ultrastructural,cytochemical and immunocytochemical investigation of the interphase nucleus in the unicellular green alga Chlamydomonas reinhardtii

Summary— The ultrastructural organization of the interphase nucleus of the green alga Chlamydomonas reinhardtii was investigated and found to be largely dependent on the fixation conditions. In specimens stained with bismuth, densely contrasted granules ranging from 25 to 45 nm in diameter were localized throughout the interchromatin space and often formed clusters. These granules were labeled by RNase A-gold complexes and may represent the counterparts of animal and higher plant cll interchromatin granules. Within the nucleolus the Ag-NOR and pyroantimonate stains and, to a lesser extent, the bismuth stain reacted with the nucleolar dense fibrillar component (DFC). When cells were subjected to a heat shock at 42°C, the nucleolar DFC was found to progressively separate from the nucleolus and, after 3 h, appeared as a continuous meandering thread about 0.1 μm in width. Within the nucleolus, labeling on conventional preparations occurred as small clusters with antibodies to H3 histones or to DNA whereas RNase A-gold complexes labeled most of it including fibrillar centers. Improved ultrastructural preservation in cryofixed, cryosubstituted specimens gently fixed in glutaraldehyde permitted to localize nucleolar DNA predominantly at the outer edge of fibrillar centers and to a lesser extent within the neighbouring DFC. Our results indicate that the structure and composition of Chlamydomonas interphase nuclei are comparable, despite particularities, to those of animal and higher plant nuclei.     

Early events of secretory granule formation in the rat parotid acinar cell under the influence of isoproterenol. An ultrastructural and lectin cytochemical study

The events involved in the maturation process of acinar secretory granules of rat parotid gland were investigated ultrastructurally and cytochemically by using a battery of four lectins [Triticum vulgaris agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), Glycine max agglutinin (SBA), Arachys hypogaea agglutinin (PNA)]. In order to facilitate the study, parotid glands were chronically stimulated with isoproterenol to induce secretion. Specimens were embedded in the Lowicryl K4M resin. The trans-Golgi network (TGN) derived secretory granules, which we refer to as immature secretory granules, were found to be intermediate structures in the biogenesis process of the secretory granules in the rat parotid acinar cell. These early structures do not seem to be the immediate precursor of the mature secretory granules: in fact, a subsequent interaction process between these early immature granule forms and TGN elements seems to occur, leading, finally, to the mature granules. These findings could explain the origin of the polymorphic subpopulations of the secretory granules in the normal acinar cells of the rat parotid gland. The lectin staining patterns were characteristic of each lectin. Immature and mature secretory granules were labelled with WGA, SBA, PNA, and lightly with UEA-I. Cis and intermediate cisternae of the Golgi apparatus were labelled with WGA, and trans cisternae with WGA and SBA.     

Chromatin organization in frog Purkinje neurons during the annual cycle: cytochemical and ultrastructural studies

Feulgen-DNA microdensitometric and propidium Iodide-DNA (PI-DNA) microfluorometric evaluations were made in Purkinje cells of cerebella from hibernating frogs and from active frogs. Higher Feulgen-DNA/distribution area ratios and lower PI-DNA values were found in the hibernating frog (both at high and low fluorochrome concentrations). These data indicate a higher degree of chromatin compactness. During the activity period, the Feulgen-DNA/distribution area ratios are lower and the Feulgen-DNA content is higher; the corresponding higher PI-DNA values indicate a lower degree of chromatin condensation. Histochemical and ultrastructural data show clearly different distribution patterns of chromatin and ribosomes in the two periods. The overall results underscore lower activity of neurons and a greater homogeneity within the Purkinje cell population during hibernation.     

Lysosomal degradation of bile deposits in cholestasis. An ultrastructural and cytochemical study.

Deposits of bile in the cytoplasm of hepatocytes from six patients with cholestasis were studied by electron microscopy and ultrastructural cytochemistry. The deposits were identified in vacuoles of varying size and complexity. The demonstration of acid phosphatase activity in these vacuoles suggests that lysosomes participate in the elimination of bile deposits retained in hepatocytes.     

Establishment and maintenance of nuclear position during zoospore formation in allomyces macrogynus: roles of the cytoskeleton

The involvement of the microtubule (MT) and actin microfilament (MF) cytoskeletons in establishing nuclear positions during zoosporogenesis in Allomyces macrogynus was assessed using selective cytoskeletal disrupting treatments and documented with light microscopy. These experiments were coupled with low-speed centrifugation studies to determine the degree to which cytoskeletal elements anchor nuclear position. At the onset of zoospore formation, nuclei were positioned only in cortical cytoplasmic regions of the zoosporangia (ZS). Immunofluorescence microscopy revealed that MTs primarily emanated from centrosomal regions into the surrounding cytoplasm at this stage. During delimitation of the cytoplasm into individual uninucleate zoospores, nuclei migrated from cortical regions to become distributed throughout the cytoplasm. Coincident with nuclear migrations, MTs were primarily organized at and emanated from nuclear surfaces, forming extensive perinuclear arrays. Nuclear migrations were suppressed in ZS induced to sporulate in the presence of cytochalasin D, an actin MF inhibiting compound. Disruption of MTs with nocodazole did not block nuclear migrations, although resultant nuclear spacing was irregular. Centrifugation treatments of control and drug-treated ZS demonstrated that nuclear positions were stabilized by perinuclear MT arrays. The results indicate that nuclear motility in ZS of A. macrogynus is the result of an actin-based system while perinuclear MTs arrays function to establish and fix nuclear position during zoospore formation. Copyright 1998 Academic Press.