Further use of fluorochromes in the cytochemical characterization of phytoplankton

Summary The rapid, specific effects of 25 fluorochromes at low concentration and physiological conditions of pH and temperature were investigated on live cells of five phytoplankton species (Prorocentrum micans, Amphidinium carterae, Dunaliella tertiolecta, Chlamydomonas moewusii andFragilaria crotonensis). They allowed the identification of cellular components such as the plasma membrane, endoplasmic reticulum, Golgi apparatus, thecal plates, nucleus, mitochondria, trichocysts, vacuoles/lysosomes, polyphosphate and starch granules, lipid bodies and hydrolytic enzymes. Morphological alterations of some of these constituents were examined in cells at different metabolic states. It was found that the thickness ofProrocentrum thecal plates increases during cell development while surface pores appear to be formed in the early stages of thecal formation. The number and size of mitochondria varies among cells at different stages of growth. The number of trichocysts, the size of vacuoles and the quantity of polyphosphates, starch or lipid inclusions increases in nitrogen-depleted cells. Photodegradation and photoenhancement phenomena are described. Some important factors helping to avoid quenching and some applications of the fluorochroming technique are presented.     

OBSERVATIONS ON THE FINE STRUCTURE OF THE CRYPTOPHYCEAE. I. THE GENUS CRYPTOMONAS1,2

The fine structure of 3 members of the genus Cryptomonas, C. rostrella, C. reticulata, and C. calceiformis, has been examined. These species exhibit the features typical of the class Cryptophyceae. The presence of subpellicular trichocysts causes a regular folding of the periplast, and larger trichocysts are present within the gullet. The plastid contains thylacoids arranged in pairs and a prominent pyrenoid; both structures are enclosed in 4 membranes, the outermost of which is a rough endoplasmic reticulum. The nucleus, Golgi apparatus and the Corps de Maupas occupy characteristic positions within the cell, and are the most characteristic features. All these structures indicate that this algal class occupies a unique phylogenetic position.     

Developmental sequence of the attack apparatus ofHaptoglossa mirabilis

Summary The most prominent ultrastructural characteristics of the cyst ofHaptoglossa mirabilis are a large centrally-placed nucleus which is partially ringed by three or four parallel cisternae of rough endoplasmic reticulum (r-ER), a centriole pair and single large Golgi complex which occupy the anterior end of the cell, and a population of provacuoles which occupies the posterior. During germination these organelles migrate into a narrow germ tube which subsequently expands to form the gun cell initial. The extracellular components of the attack apparatus (i.e. missile and injection tube) are formed entirely in the developing gun cell; indirect evidence suggests that both the Golgi complex and r-ER are involved in their synthesis. The intra-cellular component of the attack apparatus comprises the posterior, anterior and apical vacuoles. The posterior vacuole forms by fusion and expansion of the original cyst provacuoles; the formation of the anterior and apical vacuoles occurs late in gun cell differentiation and involves fusion of Golgi-derived vesicles.     

Secretory proteins in a ciliate cell: Identification of epitopes common to numerous polypeptides

Secretory vesicles of the ciliate Pseudomicrothorax dubius, called trichocysts, are separated into > 40 proteins by two-dimensional gel electrophoresis. The trichocyst, composed of a shaft and four arms, is in a condensed state when docked in the cell cortex, and it elongates into an extended state during exocytosis. Monoclonal antibodies (mAbs) were raised against trichocyst proteins. Their reactivities were analysed: I) on Western blots of extended, isolated trichocysts by immunolabeling; 2) on entire cells and extended trichocysts by indirect immunofluorescent binding assay (IFA); 3) on semi-thin sectioned cells by IFA; and 4) on ultra-thin sections of cells by immunogold labeling. mAb IV 4E5 labels major trichocyst proteins at 15–19, 22 and 24 kDa, pI 4.6?6.6. The epitope recognized by mAb IV 4E5 is common to as many as 30 proteins and suggests a family of proteins with possible sequence homology. By IFA, the shafts of extended trichocysts are labeled. The shafts of condensed trichocysts are labeled on both semi-thin sections in Lowicryl and ultrathin sections. On semi-thin Epon sections, the part of the trichocyst which is labeled is arm-like. mAb VI 2D12 labels three major trichocyst proteins at 31 kDa, pI 5.0?5.4. The arms of extended trichocysts are labeled by IFA, but are only weakly labeled on ultrathin sections. The shaft of extended trichocysts is labeled by IFA, and the shaft of condensed trichocysts is labeled on ultrathin sections.     

The Trichocysts of Chilomonas paramecium*

SYNOPSIS Axenic cultures of Chilomonas paramecium were grown in media lacking a C-source, resulting in breakdown in autophagosomal vesicles of large numbers of trichocysts. Return of the starved organisms to complete media was followed by a wave of trichocyst formation. Stages in the degeneration and subsequent reformation of trichocysts are described as well as attempted labeling of the developing organelles with ³H-thymidine. A modification of the method of Anderson et al. (2) was used for isolating quantities of exploded trichocysts from Chilomonas. Attempts at isolation of the trichocyst in its coiled state were unsuccessful. Isolated trichocysts mounted on electron microscope grids were subjected to various types of enzymatic digestions.     

Fine structure of the sweat glands of the antebrachial organ of Lemur catta

Summary The sweat glands of the antebrachial organ of the ring-tailed lemur are atypical apocrine glands which have some characteristics of eccrine sweat glands. The myoepithelial cells are large and consist of well-differentiated basal and apical regions. The secretory cells form a monolayer of tall, columnar cells filled with numerous secretory vacuoles and capped with differentiated apical blebs. The vacuoles are formed in the Golgi region and their contents are discharged into the lumen and into intercellular canaliculi. The blebs are pinched off at the luminal surface by a true apocrine mechanism. In addition to the usual organelles (abundant rough endoplasmic reticulum, prominent Golgi region, large mitochondria, pigment, secretory vacuoles), the secretory cells contain bundles of microtubules. Each microtubule is about 325–350 Å in diameter. The glands are larger and more active in the male. These sweat glands are distinctly different from the apocrine glands of the general body surface of L. catta.Publication No. 128 of the Oregon Regional Primate Research Center, supported in part by Grants FR 00163 and AM 08445 from the National Institutes of Health. The author expresses thanks to D. McLean for preparation of the diagram.     

An electron microscopical study of absorbing cells in the posterior caput epididymidis of rabbits

Summary The columnar cells in regions 3 and 4 of the ductus epididymidis in rabbits display ultrastructural features characteristic of absorbing cells. The stereocilia show basal anastomoses and often a fibrillar core continuous with a fibrillar web in the apical cytoplasm. Numerous invaginations of the slightly downy apical cell membrane and many thick-walled apical vesicles and vacuoles contain an opaque substance similar to that seen in the lumen. The vacuoles often contain small vesicles or bodies, probably formed from the vacuolar wall by budding. Numerous bodies or vacuoles with moderately dense contents are seen in the Golgi area and in the supranuclear and intranuclear cytoplasm in region 3. In region 4 they are denser and mainly seen above the nucleus. A high acid phosphatase activity was demonstrated in most dense and some light bodies. India ink introduced by way of the rete testis was taken up from the lumen into apical invaginations, vesicles and vacuoles and slowly transferred to denser bodies below the Golgi apparatus.These observations are interpreted as evidence for a resorption of substances from the lumen by a pinocytotic process, and for their storage and perhaps digestion in the dense bodies, which appear to have a lysosomal character. The Golgi apparatus is large with many vesicles of two types and empty cisternae but few typical Golgi vacuoles. The partly granular endoplasmic reticulum is very well developed and has opaque contents. Microtubules run from the terminal bar region into the Golgi area. Thick-walled vesicles occur throughout the cytoplasm, sometimes in continuity with the cell membrane. The basal parts of the cell borders often interdigitate.Supported by a grant from the Swedish State Medical Research Council.     

Filamentous actin in paramecium cells: functional and structural changes correlated with phalloidin affinity labeling in vivo

Rhodaminylated (R)-phalloidin microinjected into Paramecium tetraurelia cells at a final concentration of greater than or equal to 20 micrograms/ml produces considerable functional and structural changes. F-actin bundles (with 20 micrograms/ml phalloidin within 15 min) are formed, which subsequently (greater than 30 min) are sequestered into autophagic vacuoles; simultaneously, the originally intense fluorescence of a narrow cortical layer becomes more and more diminished. When such microinjected cells are processed for electron microscopy, they display concomitant ultrastructural alterations, namely, the formation of transcellular bundles of 5-7 nm-thick filaments, which subsequently appear in autophagosomes, as well as a considerable reduction of filamentous materials in the cortex. This, in turn, entails a considerable restructuring of the cortex, enabling free access of various structural components to the cortex. Higher doses of R-phalloidin abolish cytoplasmic streaming (e.g., 50 micrograms/ml after 20-30 min); although the cells may survive, new secretory organelles (trichocysts) are no longer docked to the cell membrane. In contrast, exocytosis of docked trichocysts (as well as subsequent membrane resealing and retrieval) is not impaired under any conditions. Cortical F-actin may account for the cytoplasmic streaming that may normally guarantee the delivery of new trichocysts to free docking sites at the cell membrane. When docking is inhibited by high R-phalloidin doses, excess free trichocysts are sequestered into autophagosomes (crinophagy). One of the most sensitive cell functions is food vacuole formation (assayed by prelabeling with India ink), which correlates with the presence of R-phalloidin labeling in the cytostomal region and around food vacuoles. The main conclusions from this work are that filamentous actin may be involved in structuring of the cortex and in cytoplasmic streaming, and may therefore influence the formation, and possibly the transcellular transport (cyclosis), of food vacuoles, as well as the docking of trichocysts, whereas it does not play a role in exocytosis per se or in the steps immediately following.     

THE ULTRASTRUCTURE OF GLENODINIOPSIS STEINII (DINOPHYCEAE)

The freshwater dinoflagellate Glenodiniopsis steinii Wolsoszyńska was examined using computer-assisted three-dimensional reconstruction of serially sectioned cells observed with the transmission electron microscope and images from the scanning electron microscope. Vegetative cells contain ultrastructure typical of freshwater dinoflagellates including trichocysts, mitochondria, Golgi bodies, starch grains, and lipid bodies. The chloroplast is a single, multilobed structure, not multiple discoid chloroplasts as previously described. The “C” shape of the nucleus is apparently due in part to the size and location of the pusule.     

AMPHIDINIUM CRYOPHILUM SP. NOV. (DINOPHYCEAE) A NEW FRESHWATER DINOFLAGELLATE. II. ULTRASTRUCTURE1

The dinoflagellate Amphidinium cryophilum sp. nov. is one of the few gymnodinians to be studied at the ultrastructural level. It resembles other dinoflagellates in the structure of the nucleus, trichocysts, storage materials, flagella, mitochondria, and microbodies. Other features of A. cryophilum less commonly observed in related organisms include a network of small interconnected vesicles, a system of large, peripheral vacuoles, chloroplasts bound by two rather than three membranes, an accumulation body, thylakoid-associated plastoglobuli, a vesiculated nuclear envelope, a complex tubular pusule, striated flagellar collars, collared pits, and a peduncle. The occurrence of a peduncle, a structure implicated in phagotrophy, in this autotrophic organism is noteworthy. The ultrastructure of the peduncle of A. cryophilum differs significantly from that reported in another dinoflagellate.     

Immunogold localization of prolamines in developingHaynaldia villosa endosperm

Summary Haynaldia villosa is a wild grass belonging to the tribe Triticeae, which includes important crops such as wheat, barley, and rye. The alcohol-soluble proteins ofH. villosa have extensive immunological relatedness with wheat prolamines as visualized by Western blot analysis. Amorphous protein inclusions surrounded by a limiting membrane are commonly found in the vacuoles of endosperm and subaleurone layers ofH. villosa seeds. A layer of cells just beneath the aleurone layer is rich in ER. Unlike that in other cell types, the ER in these cells is highly dilated and contains materials at its swollen distal ends. These materials are structurally similar to substances found in the protein bodies. Protein A-gold immunocytochemical localization studies employing antibodies against wheat prolamine confirmed that the inclusions found in the lumen of the ER do not contain prolamines. This observation indicates that the ER does not act as the site of prolamine accumulation inH. villosa. Protein bodies found in the vacuoles and the vesicles associated with the Golgi complexes were specifically labeled. This suggests that Golgi complexes mediate the transport of prolamines into vacuoles ofH. villosa endosperm cells, in a fashion analogous to that of other vacuolar proteins of dicotyledonous plants.     

Observations on the ultrastructure of representatives of the genera Hemiselmis and Chroomonas (Cryptophyceae)

Hemiselmis rufescens Parke and three species of the genus Chroomonas have been examined by electron microscopy. They demonstrate certain characteristic features of the Cryptophyceae, such as the presence of trichocysts and the arrangement of thylakoids in pairs in the plastid. The prominent pyrenoid of the genus Chroomonas is penetrated longitudinally by a tongue of cytoplasmic matrix which originates from between the two pairs of plastid membranes. In the genus Hemiselmis, however, the pyrenoid is traversed by a pair of thylakoids. The nucleus, Golgi apparatus and Corps de Maupas also occupy characteristic positions. These features indicate a close relationship between these two genera and the other major genus Cryptomonas, and support the suggestion that the Cryptophyceae is a discrete taxonomic group.     

Fine Structure of Silica Deposition and the Origin of Shell Components in a Testate Amoeba Netzelia tuberculata1

ABSTRACT Netzelia tuberculata secretes a test composed of siliceous particles cemented together by organic plaques forming a single-layered spheroidal shell. The siliceous particles are produced within cytoplasmic vacuoles by three mechanisms: 1) synthesis de novo by deposition of the silica on a matrix; 2) deposition of silica on particles remaining in digestive vacuoles, including starch grains and undigested walls of yeast cells; and 3) secretion of silica as a hollow sphere at the periphery of vacuoles enclosed by the silicasecreting membrane. The silicalemma (silica-secreting membrane) originates as fibril-containing vesicles (GFV) secreted by the Golgi body. Fusion of these vesicles with membranes surrounding digestive vacuoles or with membranes surrounding specialized vacuoles containing a silica-binding matrix apparently converts the vacuole into a silica-depositing organelle. Small spherules of silica occur on the vacuolar side of the membrane surrounding the developing test granules, marking the presence of silicalemma activity. These colloidal spherules become aggregated into larger spherules that condense to form the siliceous surface of the developing test particle. Other Golgi vesicles, designated Golgi plaque vesicles (GPV), produce the organic plaques that are deposited among the siliceous particles at the periphery of the cell during new test construction during cell division. The fine structure of the GFV and GPV and their role in test wall deposition are discussed in relation to other silica-biomineralizing protozoa, including radiolaria.     

The function of the golgi complex in transitional epithelium. Synthesis of the thick cell membrane

The superficial squamous cells of rat transitional epithelium are limited, on their luminal face, by an asymmetrically thickened membrane. Patches of similar thick membrane are found in the walls of the Golgi cisternae and it is suggested that the Golgi system is the site of assembly of the thick plasma membrane. This implies membrane flow from the Golgi apparatus to the cell surface, and there is indirect evidence that the membrane is transported in the form of fusiform vacuoles, derived from the Golgi cisternae, which fuse with, and become part of, the free cell membrane. Uptake of injected Imferon shows that similar, large, thick-walled vacuoles may be formed by invagination of the free cell surface. Some of these vacuoles are subsequently transformed into multivesicular bodies and autophagic vacuoles. The formation of other large heterogeneous bodies is described, and some of these are shown to have acid phosphatase activity.     

ULTRASTRUCTURAL OBSERVATIONS ON THE TRICHOBLASTS OF EQUISETUM

Equisetum trichoblasts are densely cytoplasmic, containing numerous starch-containing plastids, mitochondria, and concentrations of rough endoplasmic reticulum with attached polysomes. Numerous vesicles of Golgi origin are present, containing a lightly staining fibrillar material; these vesicles appear to fuse with the wall. The outer tangential and radial walls become thickened while the inner tangential wall remains thin with numerous plasmodesmata. As the trichoblasts develop into root hairs, vacuolation occurs, resulting in large vacuoles. This may represent autolytic vacuolation. The cytoplasm of the root hairs is similar to that of the trichoblasts.     

The Terminal Protonephridial Complex of Haplopharynx rostratus (Platyhelminthes,Haplopharyngida)

The terminal protonephridial complex of Haplopharynx rostratus consists of three terminal cells. There are no weirs consisting of ribs connected by a filtration “membrane”, but some cytoplasmic outgrowths into the lumen of the terminal cells. Excretion is by exocytotic vesicles. The terminal cells also contain Golgi complexes and large membrane-bound vacuoles containing electron-dense material. The ciliary bundles (flames) of terminal cells 2 and 3 protrude into the lumen of the centrally located terminal cell I. The complex is surrounded by a sheath containing numerous filaments. The terminal complex of H. rostratus resembles that of the macrostomid Paromalostomum proceracauda, lending support to the view that the two taxa are closely related. © 1998 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd. All rights reserved     

Protein Uptake and Digestion in Bloodstream and Culture Forms of Trypanosoma brucei*

SYNOPSIS. The mechanisms of ferritin uptake and digestion differ in bloodstream and culture forms of Trypanosoma brucei. Ferritin enters bloodstream forms from the flagellar pocket by pinocytosis in large spiny-coated vesicles. These vesicles become continuous with straight tubular extensions of a complex, mostly tubular, collecting membrane system where ferritin is concentrated. From the collecting membrane system the tracer enters large digestive vacuoles. Small spiny-coated vesicles, which never contain ferritin, are found in the Golgi region, fusing with the collecting membrane system, and around the flagellar pocket. Acid phosphatase activity is present in some small spiny-coated vesicles which may represent primary lysosomes. This enzymic activity is also found in the flagellar pocket, pinocytotic vesicles, the collecting membrane system, the Golgi (mature face), and digestive vacuoles of bloodstream forms. About 50% of the acid phosphatase activity of blood forms is latent. The remaining nonlatent activity is firmly cell-associated and probably represents activity in the flagellar pocket. The structures involved in ferritin uptake and digestion are larger and more active in the short stumpy than in the long slender bloodstream forms. The short stumpy forms also have more autophagic vacuoles. No pinocytotic large, spiny-coated vesicles or Golgi-derived, small spiny-coated vesicles are seen in culture forms. Ferritin leaves the flagellar pocket of these forms and enters small smooth cisternae located just beneath bulges in the pocket membrane. The tracer then passes through a cisternal collecting membrane network, where it is concentrated, and then into multivesicular bodies. In the culture forms, acid phosphatase activity is localized in the cisternal system, multivesicular bodies, the Golgi (mature face), and small vesicles in the Golgi and cisternal regions. The flagellar pocket has no acid phosphatase activity, and almost all the activity is latent in these forms. The culture forms do not release acid phosphatase into culture medium during 4 days growth. Uptake of ferritin by all forms is almost completely inhibited by low temperature. These differences among the long slender and short stumpy bloodstream forms and culture forms are undoubtedly adaptive and reflect different needs of the parasite in different life cycle stages.     

STRUCTURE AND REPRODUCTION OF THE ALGAL SYMBIONTS OF HYDRA VIRIDIS1

The algal symbionts of Hydra viridis are found within vacuoles of the gastrodermal digestive cells of the host. Electron microscopy reveals that the symbionts possess cell walls, and that their reproductive cycle follows the general pattern of free-living Chlorella. Nuclear and chloroplast divisions arc followed by formation of new cell walls, the Golgi apparatus being quite active during cell wall synthesis. Autospores are released when the parent wall ruptures. The autospores are then usually segregated into separate animal vacuoles. Remnants of the ruptured parent wall persist in the vacuoles for an indefinite period. The ruptured parent walls curl at the breakage clefts, forming double-layered scroll-like structures. The fate of these wall remnants has not been firmly established. Long-term starvation of the animals does not result in a detectable change in the structure of the symbionts, and they continue to divide and to store carbohydrate as starch grains.     

THE PRESENCE OF PROTEASE-DIGESTIBLE MATERIAL IN GOLGI VESICLES DURING ENDOSPERM DEVELOPMENT OF SELECTED CEREALS

The presence of dictyosomes secreting densely stained vesicles throughout endosperm protein body formation was confirmed for four cereals (rice, Oryza sativa L.; hard red winter wheat, Triticum aestivum L.; winter feed barley and spring malting barley, Hordeum vulgare L.; oats, Avena sativa L.). The contents of the Golgi vesicles and protein bodies were digested with proteases for all cereals except rice. It was found in the case of rice that OsO₄ altered the proteins in the Golgi apparatus and protein bodies making them resistant to protease digestion. These results imply that the Golgi apparatus plays an important role in the concentration and transport of storage proteins into vacuoles.     

ULTRASTRUCTURE AND DEVELOPMENT OF THE INACTIVE STOMATA OF ANABASIS ARTICULATA (FORSK.) MOQ.

The guard cells of Anabasis articulata mature and senesce a short distance from the intercalary meristem in which they form. When the guard cells reach final size, their ultrastructure is similar to that of stomata of other plants. At this stage, they contain clearly definable, numerous mitochondrial profiles, chloroplasts with starch grains and plastoglobuli, active Golgi bodies, a large nucleus that stains deeply for chromatin and large vacuoles. During later stages of development the whole protoplasmic content becomes very dense, with myelin-like figures and crystals appearing in the vacuoles. The cell walls thicken considerably. This is especially true of the tangential walls, where the microfibrils of different lamellae vary in their orientation. It is suggested that as a result of these ultrastructural changes the guard cells lose the ability to move.