ANALYSIS OF MUCILAGE SECRETION AND EXCRETION IN MICRASTERIAS (CHLOROPHYTA) BY MEANS OF IMMUNOELECTRON MICROSCOPY AND DIGITAL TIME LAPSE VIDEO MICROSCOPY1

Two different, independent, and alternative modes of mucilage excretion were found in the unicellular green alga Micrasterias denticulata Bréb. under constant culture conditions. The cells were capable of either excreting mucilage over all their cell surface or they extruded mucilage from one of their polar ends, which enabled directed movement such as photoorientation or escape from unfavorable environmental conditions. By means of a polyclonal antibody raised against Micrasterias mucilage, the secretory pathway of Golgi derived mucilage vesicles from their origin to their discharge was analyzed by means of conventional and energy filtering TEM. Depending on the stage of the cell cycle, mucilage vesicles were subjected to maturation processes. This may occur either after they have been pinched off from the dictyosomes (e.g. during cell growth) or when still connected to trans‐Golgi cisternae, as in the case of interphase cells. Only fully grown mature vesicles contained mucilage in its final composition as indicated by antibody labeling. After fusion of mucilage vesicles with vacuoles, no immunolabeling was found in vacuoles, indicating that the vesicle content was digested. Mucilage vesicles fused with the plasma membrane in areas of cell wall pores but were also able to excrete mucilage at any site directly through the respective cell wall layer. This result disproves earlier assumptions that the pore apparatus in desmids are the only mucilage excreting areas at the cell surface. Both mechanisms, excretion through the pores and through the cell wall, lead to formation of mucilage envelopes covering the entire cell surface.     

Improved preservation of the form and contents of wall vesicles and the golgi apparatus in freeze substituted hyphae ofSaprolegnia

Summary Secretory vesicles involved in cell wall synthesis (wall vesicles) and the Golgi apparatus have been compared in conventionally fixed and freeze substituted hyphae of the oomycete fungusSaprolegnia ferax. Wall vesicles freeze substituted in various fluids range from spherical to tubular and contain an intensely staining, phosphorous rich matrix. In contrast diverse conventional fixations cause artefactual constrictions in most tubular vesicles and loss of their intensely staining contents. These data are interpreted to show the existence of an intravesicular skeletal system, with cellular regulation, to determine vesicle morphology and intravesicular synthesis of a hypothetical phosphorylated glycolipid cell wall precursor. Whilst freeze substitution gives superior preservation of wall vesicle morphology, it does not demonstrate any preferential association between wall vesicles and microtubules thus suggesting that microtubules are only indirectly involved in wall vesicle transport. Freeze substitution is superior to conventional fixation for analysis of the Golgi apparatus because it uniquely reveals both differentiation of a specific single cisterna in each Golgi body and greater differences in membrane thicknesses throughout the endomembrane system.     

Ultrastructural Studies of a Vesicle System Associated with Endoplasmic Reticulum in Exo-Erythrocytic Forms of Plasmodium berghei1

ABSTRACT. Fine structural studies of a specialized vesicle system associated with the endoplasmic reticulum (ER) of exo-erythrocytic Plasmodium berghei suggest that this system may be the equivalent of a Golgi apparatus. Patches of ER, randomly distributed in the cytoplasm of developing parasites, are formed of smooth and ribosome-studded cisternae intermingled with each other. The vesicle systems are located between as well as at the edges of ER aggregates and appear to be in different stages of budding from the cisternae. Prolonged osmication reveals distinct staining of the nuclear envelope and ER of the parasites as well as part of the Golgi apparatus of the hepatocytes. However, the small vesicles associated with the parasite's ER are unstained, as are the coated vesicles in the Golgi region of the liver cell. These sites in the parasite cytoplasm seem comparable to the concave surface of the Golgi apparatus in liver cells. The pinched-off vesicles fuse with others to form the prominent peripheral vacuolization characteristic of the nearly mature exo-erythrocytic form. The formation of these peripheral vacuoles and their subsequent fusion with the parasite membrane may be an exocytosis mechanism supplying the rapidly expanding parasite with new plasma membrane material.     

Cell-free analysis of Golgi apparatus membrane traffic in rat liver

 Cell-free systems for the analysis of Golgi apparatus membrane traffic rely either on highly purified cell fractions or analysis by specific trafficking markers or both. Our work has employed a cell-free transfer system from rat liver based on purified fractions. Transfer of any constituent present in the donor fraction that can be labeled (protein, phospholipid, neutral lipid, sterol, or glycoconjugate) may be investigated in a manner not requiring a processing assay. Transition vesicles were purified and Golgi apparatus cisternae were subfractionated by means of preparative free-flow electrophoresis. Using these transition vesicles and Golgi apparatus subfractions, transfer between transitional endoplasmic reticulum and cis Golgi apparatus was investigated and the process subdivided into vesicle formation and vesicle fusion steps. In liver, vesicle formation exhibited both ATP-independent and ATP-dependent components whereas vesicle fusion was ATP-independent. The ATP-dependent component of transfer was donor and acceptor specific and appeared to be largely unidirectional, i.e., ATP-dependent retrograde (cis Golgi apparatus to transitional endoplasmic reticulum) traffic was not observed. ATP-dependent transfer in the liver system and coatomer-driven ATP-independent transfer in more refined yeast and cultured cell systems are compared and discussed in regard to the liver system. A model mechanism developed for ATP-dependent budding is proposed where a retinol-stimulated and brefeldin A-inhibited NADH protein disulfide oxidoreductase (NADH oxidase) with protein disulfide-thiol interchange activity and an ATP-requiring protein capable of driving physical membrane displacement are involved. It has been suggested that this mechanism drives both the cell enlargement and the vesicle budding that may be associated with the dynamic flow of membranes along the endoplasmic reticulum-vesicle-Golgi apparatus-plasma membrane pathway. Accepted: 26 January 1998     

Ultrastructure of meristematic epidermal cells of maize root under water deficit conditions

Summary Structural components of meristematicZea mays primary root epidermal cells were observed after osmotic stress of the nutrient medium (12.5 atm.) and after rehydration. After non-lethal osmotic stress (24 hours), aggregation of nuclear chromatin, parallel ER arrangement, and reduction of mitochondrial cristae were found. Increased number of Golgi cisternae indicated vesicle production inhibition, and microtubules were absent in treated cells, although plastid structure remained unchanged. After lethal osmotic stress (48 hours), fragmentation of cytoplasmic membranes and a more severe structure damage of all cellular components occurred. Structure of the nucleus, mitochondria, Golgi apparatus and microtubules reappeared in the cells after rehydration. The only new feature of these cells was occurrence of smooth ER, which may indicate that the ER system has acquired a different function in regenerated cells.     

MUCILAGE PROCESSING AND SECRETION IN THE GREEN ALGA CLOSTERIUM. II. ULTRASTRUCTURE AND IMMUNOCYTOCHEMISTRY1

We conducted an ultrastructural and immunocytochemical analysis of the subcellular components involved in mucilage secretion in Closterium. In conventionally fixed cells, the mucilage vesicle appears dense-cored with an electron-dense center surrounded by radiating fibrils. In freeze-substituted cells, the vesicles are highly osmiophilic. These mucilage vesicles are produced from peripheral swellings of the trans face cisternae of the Golgi apparatus (GA). The vesicles apparently move from the GA, found in cytoplasmic depressions between lobes of the plastid, to the sub-plasma membrane peripheral cytoplasm. Here, they become associated with components of the peripheral cytoskeletal network. The mucilage is ultimately released through flask-shaped pores in the cell wall.     

Vesicle production and fusion during lobe formation inMicrasterias visualized by high-pressure freeze fixation

Summary Two different types of Golgi vesicles involved in wall formation can be visualized during lobe growth inMicrasterias when using high-pressure freeze fixation followed by freeze substitution. One type that corresponds to the dark vesicles (DV) described in literature seems to arise by a developmental process occurring at the Golgi bodies with the single vesicles being forwarded from one cisterna to the next. The other vesicle type appears to be produced at thetrans Golgi network without any visible precursors at the Golgi cisternae. A third type of vesicle, produced by median andtrans cisternae, contains slime; these are considerably larger than those previously mentioned and they do not participate in wall formation. The distribution of the two types of cell wall vesicles at the cell periphery and their fusion with the plasma membrane are shown for the first time, since chemical fixation is too slow to preserve a sufficient number of vesicles in the cortical cytoplasm. The results indicate that fusions of both types of vesicles with the plasma membrane are possible all over the entire surface of the growing half cell. However, the DVs are much more concentrated at the growing lobes, where they form queues several vesicles deep behind zones on the plasma membrane thought to be specific fusion sites. The structural observations reveal that the regions of enhanced vesicle fusion correspond in general to the sites of calcium accumulation determined in earlier studies. By virtue of the absence of the DVs in the region of cell wall indentations the second type of wall forming vesicle appears prominent; they too fuse with the plasma membrane and discharge their contents to the wall.     

Micrasterias cells as a model system for research on morphogenesis.

Micrasterias species have been the subject of numerous experimental studies on cell shape formation in the last 40 years. Chemical and physical treatment during different developmental stages, as well as investigations of ultrastructure by means of various different preparation methods, have yielded information about some principles of morphogenesis in the symmetric, highly ornamented Micrasterias cell. The basic symmetry of a Micrasterias cell is determined prior to mitosis and is established without nuclear control thereafter. Normal cell development, however, may occur only under the conditions of continuous protein synthesis throughout the cell cycle. A prepattern for the later cell shape seems to be present at the plasma membrane at the early stages of septum formation. It is realized by a local, patterned distributed incorporation of cell wall material that is delivered by Golgi-produced vesicles. The areas where fusions take place between the primary wall material containing vesicles and the plasma membrane are defined by inward ionic currents that are carried at least in part by calcium. These areas develop into lobes during the following course of cell growth. Cell shaping in Micrasterias cells is thus mediated by both an enhanced extension of the cell wall and an additional incorporation of wall material in the areas of the lobes. Numerous studies have indicated that actin plays an important role in morphogenesis, whereas microtubules do not participate in this process but are involved mainly in nuclear migration. The present review shows that although a wealth of details concerning Micrasterias morphogenesis has already been elucidated, two main questions, i.e., the method of septum formation and the splitting of the lobes, remain to be answered.     

Three classes of spiny-(Clathrin-) coated vesicles in rodent liver based on size distributions

Summary Clathrin-coated vesicles in rodent (rat and mouse) liver distribute into three distinct populations based on measurements of vesicle diameter. The first population consists of 60–80 nm vesicles found almost exclusively within the Golgi apparatus region. The second population is of 100–160 nm coated vesicles located within 100–500 nm of the cell surface. A third population of coated vesicles of intermediate diameter (ca. 90 nm) is present both at the Golgi apparatus and at the cell surface. We speculate that clathrin and clathrin-coated vesicles within the region of the Golgi apparatus and of the cell surface exist in two recycling populations. The third population of vesicles of intermediate diameter could represent a shuttle to link the two major compartments.     

Observations on the origin of two substances produced by the vitellaria in Gorgoderina attenuata

The vitelline cells in Gorgoderina attenuata produce two qualitatively distinct substances. One substance assumes the form of individual, dense, osmiophilic globules. Many globules are contained in a single vesicle. The other substance is an amorphous mass of medium density that completely fills a vesicle. Observations indicate that the dense, osmiophilic globules develop in association with a system of small, contiguous, ribosome-free vesicles. It is suggested that this system of vesicles constitute a Golgi apparatus for these cells. The amorphous mass substance develops in vesicles which appear to be derived from endoplasmic reticulum. Close association between the amorphous mass vesicle and mitochondria are commonly observed.     

Transport of rosettes from the golgi apparatus to the plasma membrane in isolated mesophyll cells ofZinnia elegans during differentiation to tracheary elements in suspension culture

Summary Rosettes of six particles have been visualized by freeze-fracture in the protoplasmic fracture (PF) faces of: a) the plasma membrane, b) Golgi cisternae, and c) Golgi-derived vesicles in mesophyll cells ofZinnia elegans that had been induced to differentiate synchronously into tracheary elements in suspension culture. These rosettes have been observed previously in the PF face of the plasma membranes of a variety of cellulose-synthesizing cells and are thought to be important in cellulose synthesis. InZinnia tracheary elements, the rosettes are localized in the membrane over regions of secondary wall thickening and are absent between thickenings. The observation of rosettes in the Golgi cisternae and vesicles suggests that the Golgi apparatus is responsible for the selective transport and exocytosis of rosettes in higher plants, as has been previously indicated in the algaMicrasterias (Giddings et al. 1980). The data presented indicate that the Golgi apparatus has a critical role in the control of cell wall deposition because it is involved not only in the synthesis and export of matrix components but also in the export of an important component of the cellulose synthesizing apparatus. The rosettes are present in the plasma membrane and Golgi vesicles throughout the enlargement of the secondary thickening, suggesting that new rosettes must be continually inserted into the membrane to achieve complete cell wall thickening.Abbreviations EF Golgi vesicles, exoplasmic fracture; the plasma membrane, extracellular fracture - PF protoplasmic fracture     

Ultrastructural studies of a vesicle system associated with endoplasmic reticulum in exo-erythrocytic forms of Plasmodium berghei

Fine structural studies of a specialized vesicle system associated with the endoplasmic reticulum (ER) of exo-erythrocytic Plasmodium berghei suggest that this system may be the equivalent of a Golgi apparatus. Patches of ER, randomly distributed in the cytoplasm of developing parasites, are formed of smooth and ribosome-studded cisternae intermingled with each other. The vesicle systems are located between as well as at the edges of ER aggregates and appear to be in different stages of budding from the cisternae. Prolonged osmication reveals distinct staining of the nuclear envelope and ER of the parasites as well as part of the Golgi apparatus of the hepatocytes. However, the small vesicles associated with the parasite's ER are unstained, as are the coated vesicles in the Golgi region of the liver cell. These sites in the parasite cytoplasm seem comparable to the concave surface of the Golgi apparatus in liver cells. The pinched-off vesicles fuse with others to form the prominent peripheral vacuolization characteristic of the nearly mature exoerythrocytic form. The formation of these peripheral vacuoles and their subsequent fusion with the parasite membrane may be an exocytosis mechanism supplying the rapidly expanding parasite with new plasma membrane material.     

Ultrastructural localization of polysaccharides and N-actetyl-D-galactosamine in the secretory pathway of green algae (Desmidiaceae)

In order to characterize the secretory pathway leading to multipolar tip growth in green algae of the family Desmidiaceae, different general polysaccharide stainings, such as the periodic acid-silver hexamine method and the periodic acid-silver proteinate method as well as different lectins specific to defined sugar residues have been employed. General polysaccharide stainings label different kinds of secretory vesicles starting from the onset of vesicle production up to their delivery into the primary cell wall, however, the discrimination of Golgi products is possible using lectins. Both gold-labelled lectins from Helix pomatia and from Glycine max with affinity to N-acetyl-D-galactosamine only produce labelling of primary wall material containing 'dark vesicles' on the ultrathin sections of high-pressure frozen and freeze-substituted Micrasterias cells, whereas other vesicle types remain unstained. 'Dark vesicles' are labelled when still attached to trans-Golgi compartments, when distributed throughout the cytoplasm or when fusing with the plasma membrane with the same staining intensity which indicates that the sugars detected by the methods used are present from the onset of visible vesicle production. Gold-labelling of N-acetyl-D-galactosamine is also observed in the primary cell wall. In control experiments the staining vanishes when ultrathin sections are pre-incubated with N-acetyl-D-galactosamine. Various other lectins with affinity to different sugar residues than N-acetyl-D-galactosamine do not produce staining of the cell wall nor of any kind of secretory vesicles. As N-acetyl-D-galactosamine is usually not present in N-linked polysaccharides the results point towards the presence of O-linked-glycoproteins in the primary cell wall of desmids.Key words: Golgi, lectins, Micrasterias, N-acetyl-D-galactosamine, secretion, vesicles.      

MUCILAGE-PRODUCING CELLS IN THE SEED COAT OF PLANTAGO OVATA: DEVELOPMENTAL FINE STRUCTURE

As the ovule of Plantago ovata matures into a seed its epidermal cells are transformed from undifferentiated parenchyma to thin-walled containers of almost pure mucilage. During this process the volume of the cells increases 60–80 fold, and the protoplast degenerates to a remnant. Rapid cell expansion begins with pollination and is accompanied by an increase in the size of the nucleus and nucleolus, a change in the random arrangement of ribosomes, a decrease in the thickness of cell walls, and synthesis of starch. Deposition of mucilage inside vacuoles and between the plasma membrane and cell wall accompanies a marked increase in the number and size of Golgi vesicles. Histochemical evidence using the thiocarbohydrazide-osmium vapor method shows polysaccharide to be present within Golgi vesicles while they are still attached to the Golgi apparatus. Mucilage deposition is associated with further cell expansion, separation of the protoplast from the cell wall, fusion of vacuoles and extra protoplasmic space, and the disappearance of starch.     

CYTOCHALASIN B INFLUENCES DICTYOSOMAL VESICLE PRODUCTION AND MORPHOGENESIS IN THE DESMID EUASTRUM1

Cytochalasin B (CB) applied to young developing cells of the desmid Euastrum oblongum Ralfs ex Ralfs, at concentrations that do not entirely inhibit cytoplasmic streaming, retarded cell growth and caused malformations of cell shape. While the basic symmetry of the cell was maintained, only the first indentations were formed and the cell body appeared to be swollen. Electron microscopic investigations revealed that vesicle production at the dictyosomes was disturbed by cytochalasin. In contrast to untreated control cells, where vesicles with electron-dense contents (“dark vesicles”) were formed during primary wall formation, vesicles pinched off by the dictyosomes during CB treatment exhibited an “empty” appearance. These vesicles, which correspond to the “dark vesicles” in size, were accumulated around the dictyosomes without being transported to the plasma membrane and were frequently connected to the trans-cisternae of the Golgi bodies. We speculate that CB may influence the transfer of products from the endoplasmic reticulum (ER) to the dictyosomes via transition vesicles, which results in a disturbed vesicle production at the Golgi bodies. CB also causes a shift in ER and dictyosome distribution. Moreover, a cortical actin system appears to be involved in the cell shaping of Euastrum. The arrangement of microtubules around the nucleus is not affected by the drug.     

Estimation of Golgi membrane flow rates in ovary glands of aptenia cordifolia using cytochalasin B

Ovary gland cells of Aptenia cordifolia were exposed to 100 micrograms/ml cytochalasin B (cyt B) for 30 or 60 min during the phase of granulocrine polysaccharide secretion. The drug caused a congestion of Golgi vesicles around the dictyosomes, probably resulting from an inhibition of the vesicle migration towards the plasma membrane. The ultrastructural feature of the Golgi apparatus in control and cyt B treated cells was analyzed using stereological methods in order to estimate the mean area of vesicular membrane produced by a single dictyosome during a 30 min period of effective cyt B action. Assuming that the rate of vesicle congestion can be equated with the rate of vesicle production, the 236 dictyosomes found to be present in the non-growing ovary gland cells form 7517 vesicles in total, or approximately 32 vesicles each within a period of 30 min. This corresponds to a membrane turnover rate of 70.4 micrometers/min (this equals approximately 10% of the total plasma membrane area per min), since the mean vesicle surface area was calculated to be 0.281 microns2. The turnover time of a single Golgi cisterna was determinated to be 7.34 min, and the average vesicle life time to be 8.86 min. Discussion focuses upon the way by which the relatively high amount of vesicular membrane material incorporated into the plasmalemma is recycled into the endomembrane system. Since a bulk membrane retrieval in the form of vesicles, as well as a bulk vesicle migration from the ER to the dictyosomes could not be observed, we suggest that a transfer of membrane subunits is involved in the maintenance of membrane equilibrium in the Golgi apparatus.     

The occurrence of the Golgi apparatus in mouse oocytes. Demonstration of thiamine pyrophosphatase activity

Location of thiamine pyrophosphatase activity as a marker of the Golgi apparatus was studied ultracytochemically in mouse oocytes with germinal vesicle (OGV), oocytes at metaphase I (OMI) and oocytes at metaphase II (OMII), and further in cells of the respective cumulus oophorus serving as comparative objects. TPPase activity in cumulus oophorus cells and in OGV was found exclusively in the Golgi apparatus. In OMI the reaction product of TPPase activity was observed in isolated smooth vesicles, and in only one case in structures identifiable as the Golgi apparatus. In OMII the occurrence of TPPase activity was also recorded in isolated smooth vesicles in cortical cytoplasm and further, exceptionally, in smooth concentrically arranged vesicles or tubules. The TPPase activity was not present in vesicular complexes. The results have shown that after the resumption of meiosis the occurrence of the reaction product of TPPase activity drops abruptly due to the reduction of the Golgi apparatus. Changes affecting the Golgi apparatus after the resumption of meiosis are related to the loss of the nucleus after the germinal vesicle breakdown.     

Membrankomplexe dictyosomaler Herkunft als „Matrizen“ für die extraplasmatische Synthese und Orientierung von Mikrofibrillen

Zusammenfassung Es wird ein neu gefundener Typ von Golgi-Vesikeln (F-Vesikeln), die während der Bildung der gemusterten Sekundärwand vonMicrasterias denticulata auftreten, eingehender beschrieben. Es wird gezeigt, daß speziell differenzierte Membranen dieser Vesikel wahrscheinlich als Matrizen für die Bildung der Sekundärwand-Mikrofibrillen fungieren.F-Vesikel entstehen in einem Prozeß der Membranreifung (Matrizen-Bildung) am distalen Pol der Dictyosomen. Von dort werden diese Vesikel, die globuläre Untereinheiten enthalten, welche reihenweise in der Vesikelmembran angeordnet sind, zur Plasmamembran transportiert (Matrizen-Transfer). Dort findet eine Inkorporation und Fusion der F-Vesikelmembran mit der Plasmamembran statt (Matrizen-Inkorporation). Diese inkorporierten und ausgebreiteten Membranareale scheinen bei der Produktion der Reihen von Mikrofibrillen von besonderer Bedeutung zu sein (Matrizen-Realisation).Die elektronenmikroskopischen Ergebnisse beiMicrasterias wurden mit jenen über die Schalenbildung bei Chrysophyceen (Pleurochrysis) verglichen. Dabei wurde eine überraschende Übereinstimmung bei der Wandbildung dieser beiden Organismen festgestellt: In beiden Fällen scheint es, daß Matrizen für die Bildung der Mikrofibrillen an den Membranen von Golgi-Vesikeln gebildet werden. BeiPleurochrysis erfolgt die Bildung der Wandelemente schon innerhalb der Golgi-Zisternen, während beiMicrasterias die Zelluloseproduktion verzögert ist (time gap) und erst nach Inkorporation der Vesikelmembran in die Plasmamembran beginnt.

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Dictyosome-Derived membrane complexes as templates for the extraplasmatic synthesis and orientation of microfibrils

Summary A newly found type of Golgi vesicles (F-vesicles), occuring during the formation of the highly patterned secondary wall ofMicrasterias denticulata is described in more detail. It is shown that specifically differentiated membranes of these vesicles are probably functional as templates for the formation of the secondary wall inMicrasterias. F-vesicles are formed in a process of membrane maturation (template formation) on the distal pole of the dictyosomes. From there these vesicles, carring globular subunits orientated in rows on the vesicle membrane, are transported to the plasma membrane (template transfer). Here an incorporation and fusion of the F-vesicle membrane with the plasma membrane occurs (template incorporation). These incorporated and exposed membrane areas seem to be involved in the production of the patterned arrays of microfibrils (template realization).The electron microscopic results inMicrasterias are compared with those described for scale formation in the chrysophycean algaPleurochrysis. A surprising relationship between the types of wall formation in these two organisms could be demonstrated: in both cases templates for the production of microfibrils seem to be formed on membranes of Golgi cisternae. InPleurochrysis the formation of wall elements takes place already within the Golgi cisterna, while inMicrasterias cellulose production is delayed (time gap) and begins just after the incorporation of the vesicle membrane into the plasma membrane.

     

Ultrastructure of the pea cotyledon Golgi apparatus: Origin of dense vesicles and the action of brefeldin A

Summary We have followed the action of brefeldin A (BFA) on the Golgi apparatus of developing pea cotyledons, the cells of which are actively engaged in the synthesis and deposition of storage proteins. The Golgi apparatus of normal cells is characterized by the presence of three different types of vesicle: smooth-surfaced secretory vesicles, dense vesicles which carry the storage proteins, and clathrin-coated vesicles (CCV). The dense vesicles originate at the cis cisternae and undergo a maturation as they pass through the Golgi stack, presumably as a result of cisternal progression. CCV bud off from dense and smooth vesicles, which may be attached to one another, at the trans pole of the Golgi apparatus. BFA eliminates the CCV and leads, initially, to an increase in the number and length of the cisternae. Dense vesicles are still to be seen, and many show an increase in diameter. Longer BFA treatments result in a trans-driven vesiculation and an accumulation of vesicles within the vicinity of single cisternae. The vesicles were sometimes seen to be connected to one another via a network of tubules. As judged by immunocytochemistry with gold-coupled legumin and vicilin antisera, some of the dilated vesicles originate directly from dense vesicles by swelling whereas others probably arise by dilation of Golgi cisternae since they possess a layer of flocculent storage proteins at their periphery. By contrast the centre of the dilated vesicles labels positively with antibodies against complex glycans, indicating that the ability to segregate storage proteins from cell wall or lytic vacuole glycoproteins is lost during extended BFA treatment. The effects of BFA are reversible when cotyledons are further incubated on Gamborg's medium for 5 h without the inhibitor.Dedicated to Professor R. Kollmann on the occasion of his 65th birthday.     

Phosphatase activities and osmium reduction in cell organelles ofMicrasterias americana

Summary Organelles in resting and growing cells ofMicrasterias americana were examined using electron microscopy after cytochemical procedures for four kinds of phosphatases, acid phosphatase (ACPase), alkaline phosphatase (ALPase), thiamine pyrophosphatase (TPPase), and inosine diphosphatase (IDPase), and osmium tetroxide reduction. Special attention was paid to activities in the Golgi apparatus.In resting cells, positive reactions for ACPase and TPPase were observed in all cisternae of the dictyosome, especially in the peripheral parts. A positive IDPase reaction was seen in one central cisterna and was frequent in the distal-most cisterna. Reduction of osmium tetroxide was seen in the proximal cisternae.In early growing cells, the dictyosomes gave positive reactions for ACPase in the proximal cisternae and the distal cisterna, while in late growing cells only in proximal cisternae. Both in early and late growing cells, the dictyosomes were positive for TPPase and IDPase in the distal cisternae and vesicles derived from the distal cisternae, and for the reduction of osmium tetroxide in the proximal cisternae. ALPase activity was detected in the growing cell wall but not in the dictyosome.