The Fine-Structural Localization of Phosphatases in Neurosecretory Cells Within the Ganglia of Certain Gastropod Snails

The neurosecretory cells in the cerebral ganglia of the snail,Planorbis trivolvis, have been examined for structural detailsand distribution of phosphatase activities. A more preliminarystudy on the localization of phosphatases in the neurosecretorycells of Helix aspersa has also been made. These neurons inboth species contain typical elementary neurosecretory granuleswhich appear to be elaborated or condensed in the Golgi saccules.Immature elementary granules are identifiable as "primary lysosomes"in that they contain acid phosphatase activity as well as nucleosidephosphatases. The mature elementary granules display no phosphataseactivity. Some saccules of the Golgi are also reactive for severalnucleoside phosphatases, including thiamine pyrophosphatase(TPPase). In Planorbis, TPPase is also present in the cisternaeof the endoplasmic reticulum. Larger, membrane bound, electron-densebodies (lipochondria) are found in close spatial associationwith the Golgi region. These also possess acid phosphatase;in addition, they contain nucleoside diphosphatases and (inPlanorbis) adenosine triphosphatase. Their content of acid phosphataseand the features of their fine structure indicate that theyare lysosomes akin to the dense bodies of vertebrate neurones. The significance and implications of these results are considered,as are other details of the neuronal and glial structure inPlanorbis.     

Fine structure of the formation and fate of the residual bodies of mouse spermatozoa with evidence for the participation of lysosomes

Routine electron microscopy in combination with subcellular localization of acid phosphatase has been employed to study the formation and fate of residual cytoplasmic bodies extruded into the tubular lumen shortly before spermiation. Prior to extrusion the spermatid cytoplasm contains lipid droplets, mitochondria, ribosomes, endoplasmic reticulum, the caudally migrated Golgi apparatus, and numerous multivesicular and multigranular bodies. These membrane-limited bodies and the Golgi zone stain heavily for acid phosphatase. Following extrusion the residual bodies undergo a series of alterations: (1) disruption of multigranular bodies with release of free granules; (2) sequestration of granules, ribosomes, and reticulum inside double-membrane-limited vacuoles derived from Golgi lamellae; (3) appearance of numerous, single-membrane-bound, cytoplasmic vacuoles; (4) fragmentation; (5) peripheral migration toward the tubular wall; and (6) phagocytosis of these migrating fragments by the Sertoli cells. The demonstration of acid phosphatase activity within free granules, the sequestering Golgi lamellae, and both classes of vacuoles suggests that initial residual body degradation occurs through lysosomal cytoplasmic autophagy.     

The Basal Level Ethylene Response is Important to the Wall and Endomembrane Structure in the Hypocotyl Cells of Etiolated Arabidopsis Seedlings

The sub-cellular events that occur during the ethylene-modulated cell elongation were characterized by examining the ultra-structure of etiolated Arabidopsis seedling hypocotyl cells. Preventing the basal level ethylene response facilitated cell elongation, and the cells exhibited wall loosening and separation phenotype. Nearby the wall separation sites were frequently associated with an increase in the cortical rough endoplasmic reticulum (rER) membranes, the presence of paramural bodies, and the circular Golgi formation. The cortical rER proliferation and circular Golgi phenotype were reverted by the protein biosynthesis inhibitor cycloheximide. The cortical rER membranes were longer when the ethylene response was prevented and shortened with elevated ethylene responses. Proteomic changes between wild type and the ethylene-insensitive mutant ethylene insensitive2 (ein2) seedling hypocotyls indicated that distinct subsets of proteins involving endomembrane trafficking, remodeling, and wall modifications were differentially expressed. FM4-64 staining supported the proteomic changes, which indicated reduced endocytosis activity with alleviation of the ethylene response. The basal level ethylene response has an important role in endomembrane trafficking, biological materials transport and maintenance of the endomembrane organization. It is possible that endomembrane alterations may partly associate with the wall modifications, though the biological significance of the alterations should be addressed in future studies.     

Ultrastructure of Papillar Cells in Brassica campestris Revealed by Liquid Helium Rapid-Freezing and Substitution-Fixation Method

The ultrastructure of papillar cells of Brassica campestrison the day of anthesis was studied by the liquid helium rapid-freezingand a substitution-fixation method, abbreviated as the RFS method.Application of the RFS method to the analysis of papillar cellsenabled us to examine clear images of these cells which havenot been observed previously. The well-developed rough and smoothendoplasmic reticulum, numerous Golgi bodies and mitochondria,various small vesicles and clathrin-coated vesicles, were presentin the cells. The numbers of Golgi bodies, as well as the numbersof cisternae of each Golgi body, increased as compared to thatin the other cells of style. Lattice-like fenestrated and flattenedcisternae were seen adjacent to the narrowest trans cisternaof the Golgi body, which had a partially coated region at itsperiphery. Many coated vesicles were observed in the vicinityof this structure and the plasma membrane. Coated areas on theplasma membrane were also observed. The ultrastructure of papillarcells on the day of anthesis indicated that they are very activesecretory cells. By using an antibody against S₈-protein andsections prepared by the RFS method, we demonstrated the distributionof S₈-protein in the cell wall of papillar cells of homozygousplants of Brassica campestris S_gS₈. (Received June 26, 1990; Accepted September 29, 1990)     

Cytochemical localization of ATPase plasma membrane and acid phosphatase by cerium-based method in a salt-adapted cell line of Pisum sativum

In order to study a possible application of cerium-based techniquesin plant cells, ATPase and acid phosphatase activities havebeen compared in two cell lines of Pisum sativum calli, onesensitive to NaCI and the other selected to be grown under salinity(85 mM NaCI). ATPase activity was unchanged and localized inthe plasma membrane of both cell lines. Acid phosphatase activitywas significantly increased in the salt-selected line and localizedin the cell walls, Golgi complex, multivesicular bodies andvacuoles. These results indicated a possible involvement ofboth activities in the maintenance of cell growth in the selectedline under saline conditions. Key words: Acid phosphatase, Pisum sativum, plasma membrane ATPase, salt stress, ultrastructure     

Acid phosphatase activity during spore differentiation of the red algaeGigartina teedii andChondria tenuissima

The acid phosphatase activity during carposporogenesis inGigartina and tetrasporogenesis inChondria was studied using the Gomori technique. During the first steps of gonimoblast maturation ofGigartina, portions of cytoplasm are ensheathed by ER cisternae with acid phosphatase activity, giving rise to autolysosomal concentric membrane bodies. In a similar way large mucilage sacs are severed. They extrude their contents in a kind of exocytosis. Multivesicular bodies, concentrically arranged cisternae and extracytoplasmic compartments, each with acid phosphatase activity, remain in young carpospores for some time, probably as remnants of the autophagocytotic and exocytotic events. The Golgi apparatus is poorly developed in gonimoblast cells and young carpospores. It becomes a prominent cell component in maturing carpospores and then participates in cell wall formation. Only some of the dictyosomal cisternae contain acid phosphatase; these are irregularly distributed in the dictyosome. — In pre- and postmeiotic tetraspore mother cells ofChondria massive lead deposits are found in the dictyosomes and in adjacent Golgi vesicles. Finer lead precipitates occur in ER cisternae, especially in those which are sequestering starch-grain-containing portions of the cytoplasm to give rise to autolysosomes. During cell cleavage, the dictyosomes aggregate. They become devoid of acid phosphatase activity with the exception of vesicles at the trans face. Later, Golgi stacks associate and have common, Gomori positively reacting, narrow cisternae at the cis face. The Golgi apparatus derived cored vesicles do not contain lead precipitates whereas the Golgi cisternae in the final stage of tetrasporogenesis show acid phosphatase activity. Variations in acid phosphatase distribution are explained in the light of current models of membrane flow.Dedicated to Univ.-Prof. DrO. Härtel on the occasion of his 80th birthday.     

Using a Semi-Aqueous Medium for the Subcellular Study of Acid Phosphatase in Wheat Aleurone Tissue

Subcellular fractionation of the aleurone tissue in ethan-1,2-diol:H₂O(80:20, v/v) provided a suitable means to study acid phosphataseactivity in the protein bodies (aleurone grains). Enzyme activityin the fraction increased 7-fold during 4 d germination. Theincrease was dependent upon the presence of the embryo, butit was not regulated by gibberellic acid, indole acetic acidor glutamine when these compounds were presented either singlyor in combination. The regulatory activities of these compoundswere restricted to an acid phosphatase located in the cell wall. Key words: Triticum aestivum, aleurone tissue, acid phosphatase, plant growth regulators     

Lysosomal Compartmentation: Ultrastructural Aspects of the Origin, Development, and Function of Vacuoles in Root Cells of Lepidium sativum

The origin and development of vacuoles in root and root capcells of Lepidium sativum was investigated at the ultrastructurallevel. The vacuoles originated as vesiculations of the ER, andacid phosphatase activity was found associated with the membranesbounding developing vacuoles. As development proceeded activityof acid phosphatase became associated with the accumulated vacuolarcontent, but was confined within these bodies in all cells exceptsome of those in the outermost layer of the cap, which werein various stages of senescence. In these cells activity ofthis enzyme appeared in the cytoplasm, associated with apparenttonoplast dissolution. These findings are discussed in the context of current hypothesesconcerning the origin and relationship of vacuoles and lysosomalcompartmentation in plant cells.     

Mucilage on the Root Surface and Root Hairs of Sorghum: Heterogeneity in Structure, Manner of Production and Site of Accumulation

Small drops of a mucilaginous character near the tip of roothairs were seen by light microscopy in several species of Sorghumand the Sorghum hybrid Vidan. Electron microscopy revealed thatthe drops are formed from at least two distinct substances,both apparently secreted from the endoplasmic reticulum. In addition, a patchy, fibrillar mucilaginous layer, also withat least two components, was found on the cell wall of the roothairs and on the outer wall of ordinary root epidermal cells.Golgi bodies as well as mitochondria take part in its production.As a rule, the mucilaginous patches are colonized by bacteria. Sorghum, root hairs, mucilage     

The use of cytoenzymological changes in some parasitic protozoa for screening the carcinogenicity of chemicals

Changes in the Golgi bodies and in hydrolytic enzymes were observed in parasitic ciliates (Nyctotheroides puytoraci) and flagellates (Opalina sudafricana and Protoopalina sp.) after the administration of a single dose of 0.5 mg beta-naphthylamine (BNA) to their host, Bufo regularis. The experiment was carried out during the host's pre-breeding season, from November to February (when only trophozoites are available); the trophozoites were examined 21 days after the injection of BNA. Use of the silver impregnation technique showed an increase in the size of the granular argentophilic Golgi bodies in all three trophozoites after administering BNA to their host. In addition, a larger number of acid phosphatase and nonspecific esterase granules was found in the endoplasm of the three parasites after the injection. The localization of acid phosphatase and nonspecific esterase was found to be similar to the distribution of the Golgi bodies in both the control and the treated preparations. The results could be useful, in that some protozoans could be employed for the quick detection of chemical carcinogenicity if changes in their hydrolytic enzyme content are used as a diagnostic feature.     

Fine Structure and Cytochemistry of the Abscission Zone Cells of Phaseolus Leaves: I. Ultrastructural Changes occurring during Abscission

The fine structure of the separation zone cells from the basalabscission zone of bean leaves has been described. Examinationof these cells revealed that fracture occurred primarily asa result of the dissolution of the middle lamellar region ofthe walls, leaving intact cells on the two newly exposed fracturesurfaces. The cytoplasm and organelles within these cells appearedquite normal and showed no signs of autolysis or senescence.A comparison of the organelle numbers in these cells with cellsfrom a similar region of a control plant revealed large increasesin the number of Golgi bodies and the amount of rough endoplasmicreticulum. The significance of this finding is discussed inrelation to the secretion of cell wall hydrolysing enzymes whichare known to be produced in these cells.     

Localization of ATPase in the endoplasmic reticulum and golgi apparatus of barley aleurone

Summary The cytochemical localization of adenosine triphosphatase (ATPase) was studied in the aleurone layer of barley (Hordeum vulgare L. cv. Himalaya). Isolated barley aleurone layers secrete numerous enzymes having acid phosphatase activity, including ATPase. The secretion of these enzymes was stimulated by incubation of the aleurone layer in gibberellic acid (GA₃). ATPase was localized using the metal-salt method in tissue incubated in CaCl₂ with and without GA₃. In sections of tissue incubated without GA₃, cytochemical staining was confined to a narrow band of cytoplasm adjacent to the starchy endosperm and to the cell wall of the innermost tier of aleurone cells. Cytochemical staining was absent from the organelles of tissues not treated with GA₃. In tissue incubated in the presence of GA₃, cytochemical staining was evident throughout the cytoplasm and cell walls of the tissue. In the cell wall, electron-dense deposits were found only in digested channels. The cell-wall matrix of GA₃-treated aleurone did not stain, indicating that it does not permit diffusion of enzyme. In the cytoplasm of GA₃-treated aleurone, all organelles except microbodies, plastids, and spherosomes stained for ATPase activity; endoplasmic reticulum (ER), Golgi apparatus, and mitochondria showed intense deposits of stain. The ER of the aleurone is a complex system made up of flattened sheets of membrane, which may be associated with both the Golgi apparatus and the plasma membrane. The dictyosome did not stain uniformly for ATPase activity; rather there was a gradation in staining of the cisternae from thecis (lightly stained) to thetrans (heavily stained) face. Vesicles associated with dictyosome cisternae also stained intensely as did the protein bodies of GA₃-treated aleurone cells.     

Cytochemical detection of phosphatase and arylsulphatase activities in the midgut of Centropages typicus (Copepod, Calanoid)

Ultrastructural study of the midgut of Calanoid Copepods revealed the presence of several cell types in all species. In a previous report we described and assigned a function to each of these cell types. In order to affirm the validity of those assignments we undertook an investigation of enzymatic activity especially of phosphatase and arylsulphatase. By cytochemical methods, alkaline phosphatase activity was detected in R-, R'-D- and B-cells, with labelling being observed on the apical plasmic membrane level in all four, and in B-cells on the pinocytotic vesicle membranes. Acid phosphatase and aryl-sulphatase activities were only detectable in B-cells; the most frequently labelled structures were located in the vacuolar system, dictyosomes and Golgi vesicles, although Golgi structures occasionally reacted to acid phosphatase. Nome of the dense bodies observed in B-cells reacted to arylsulphatase. Similarly they were unevenly labelled during acid phosphatase tests. Hence it may be assumed that dense bodies are not involved in hydrolases. It is possible that these enzymes originated from vesicles generated by the Golgi saccules surrounding and joined to the vacuoles, thus bypassing the lysosome I stage.     

Ultrastructural Localization of ATPase Activity in the Cells of the Apical Meristem Zone,Elongation Zone and Root Hair Zone of Tomato Roots

A comparative study on the cytochemical localization of adenosine triphosphatase (ATPase) activity reaction in the cells of the apical meristem zone, elongation zone and root hair zone of tomato roots was carried out by electron microscopic observations of lead phosphate precipitation. The following experimental results have been obtained: In the meristematic cells of tomato roots, the heavy lead phosphate deposits indicating a very high activity of ATPase were localized at plasmalemma, plasmodesmata, endoplasmic reticulum, Golgi bodies, nucleoli and chromatin (Figs. 1—2). The reaction products of ATPase activity were also observed at some sites of ground cytoplasm and cell wall, but they were not found in little vacuoles and on tonoplast. In the cells of elongation zone, the ATPase activity at plasmalemma and plasmodesmata was as high as that in the meristematic cells of root tip, while the ATPase activity at nucleoli, chromatin, endoplasmic reticulum and Golgi bodies was markedly lowered. On the other hand, the high ATPase activity was produced on the tonoplast of the developing and enlarging vacuoles (Fig. 3). In the cells of root hair zone, the high ATPase activity was shown at plasmalemma, tonoplast and intercellular spaces, but the ATPase activity at nucleoli, chromatin and endoplasmic reticulum was wholly inactivated. (Figs. 4—7). The above results indicate that the ATPase activity with membranes and organelles is altered when the functions of cells and organelles change. Therefore, it is evident that the ATPase activity may be closely related to many physiological functions.     

Properties and biosynthesis of cell wall-bound acid phosphatase in Aspergillus nigerx

Acid phosphatase (EC 3.1.3.2 [EC] ) of Aspergillus niger myceliumwas distributed exclusively in the cell wall and soluble fractions,whereas alkaline phosphatase was distributed in the solubleand particulate fractions but only slightly in the cell wallfraction. Cell wall-bound acid phosphatase was released by fungal-walllytic enzymes such as snail gut juice. Cell wall-bound, released,and soluble acid phosphatases showed very similar enzymaticproperties except that the bound enzyme was more stable to heatand detergents. By DEAE-cellulose chromatography, the releasedacid phosphatase was found to correspond to acid phosphatasesI A, IB and II in the soluble fraction. When phosphate in the medium was consumed, the acid phosphataseactivity of the soluble fraction increased more rapidly thanthat of the cell wall fraction. When phosphate was added tothe derepressed culture, the acid phosphatase activity of thesoluble fraction decreased after a short lag period, while thatof the cell wall fraction continued to increase. When labeledamino acid was added to the derepressed culture, it was incorporatedinto the soluble acid phosphatase without a lag period, whileit was incorporated into the cell wall phosphatase after a lagperiod. From these observations, acid phosphatase was consideredto be synthesized first as the soluble form and then integratedinto the cell wall. ¹ The present experiments were carried out, for the most part,at the Institute of Applied Microbiology of the University ofTokyo. (Received January 19, 1976; )     

DISTRIBUTION OF PHOSPHATASES IN THE GOLGI REGION AND ASSOCIATED STRUCTURES OF THE THORACIC GANGLIONIC NEURONS IN THE GRASSHOPPER, MELANOPLUS DIFFERENTIALIS

The neuronal perikarya of the grasshopper contain sudanophilic lipochondria which exhibit an affinity for vital dyes. These lipochondria are membrane-delimited and display acid phosphatase activity; hence they correspond to lysosomes. Unlike those of most vertebrates, these lysosomes also hydrolyze thiamine pyrophosphate and adenosine triphosphate. Like vertebrate lysosomal "dense bodies," they are electron-opaque and contain granular, vesicular, or lamellar material. Along with several types of smaller dense bodies, they are found in close spatial association with the Golgi apparatus. The Golgi complexes are frequently arranged in concentric configurations within which these dense bodies lie. Some of the smaller dense bodies often lie close to or in association with the periphery of dense multivesicular bodies. Further, bodies occur that display gradations in structure between these multivesicular bodies and the dense lysosomes. Acid phosphatase activity is present in the small as well as the larger dense bodies, in the multivesicular bodies, and in some of the Golgi saccules, associated vesicles, and fenestrated membranes; thiamine pyrophosphatase is found in both the dense bodies and parts of the Golgi complex. The close spatial association of these organelles, together with their enzymatic similarities, suggests the existence of a functional or developmental relationship between them.     

A cytochemical study of the Golgi apparatus of the spermatid during spermiogenesis in the rat

The reactivity of the various components of the Golgi apparatus of rat spermatids for three phosphatase activities (nicotinamide adenine dinucleotide phosphatase, NADPase; thiamine pyrophosphatase, TPPase; cytidine monophosphatase, CMPase) and the incorporation of 3H-fucose by the spermatids was analyzed at the 19 steps of spermiogenesis, i.e., during and after this organelle elaborated the glycoprotein-rich acrosomic system. During steps 1-3, the Golgi apparatus produced, in addition to the proacrosomic granules, multivesicular bodies that became associated with the chromatoid body. NADPase was located within the four of five intermediate saccules of Golgi stacks, and TPPase was found in the last one or two saccules on the trans aspect of the stacks from steps 1 to 17 of spermiogenesis. CMPase was located within the thick saccular GERL elements found in the trans region of the Golgi apparatus from steps 1 to 7 of spermiogenesis, but the CMPase-positive GERL disappeared from the Golgi apparatus after its detachment from the acrosomic system at step 8. Th acrosomic system itself was reactive from CMPase and TPPase but was negative for NADPase, while the multivesicular bodies were CMPase and NADPase positive but unreactive for TPPase. Tritiated-fucose was readily incorporated within the Golgi apparatus of steps 1-17 spermatids; in steps 1-7 it was subsequently incorporated within the acrosomic system and multivesicular bodies. These various data indicated (1) that the Golgi apparatus of spermatids, although it loses its CMPase-positive GERL element in step 8, retains evidence of functional capacity until it degenerates in step 17; (2) that in early spermatids the various saccular components of the Golgi are specialized with respect to enzymatic activities; and (3) that each Golgi region may contribute in a coordinated fashion to the formation of the acrosomic system and multivesicular bodies.     

The effects of brefeldin A upon the Golgi apparatus of the green algal flagellate, Gloeomonas kupfferi

The fungal-derived derivative, brefeldin A, was used to disruptthe Golgi apparatus (GA) of the green alga, Gloeomonas kupfferi.Upon short treatments (10 µg ml^–1 for 10 min orless), the Golgi bodies maintain their perinuclear positioning.However, the medial locus transforms from a tight stack of elongatecisternae to a network of swollen tubules. Upon longer treatments(60 min), swelling and vesiculation of cis face cisternae becomeapparent. Likewise, the edges of several trans face cisternaemay fuse with those of adjacent Golgi bodies leading to theformation of multiGolgi complexes. Key words: Brefeldin A, Golgi apparatus, Gloeomonas kupfferi     

Electron microscopic and cytochemical studies of rat aorta. Intracellular vesicles containing elastin- and collagen-like material

Synopsis Small, rounded vesicles with a dense core of amorphous material were observed in all cell types in the young rat aorta, that is, endothelial cells, smooth muscle cells and fibroblasts. They were particularly numerous in the Golgi complex but were also found in the cell periphery. The content of the vesicles had staining characteristics identical to those of elastin. Material of the same type was also found in cisternae on the maturing side of the dictyosomes and in vesicles budding from them. Reaction product for thiamine pyrophosphatase was present in both these structures, indicating that the Golgi complex is responsible for the formation of the dense-cored vesicles. This was further supported by the absence of reaction product for acid phosphatase in the cisternae and in the vesicles. Moreover, no uptake of exogenous markers was noted in the latter. On the basis of these findings it is suggested that the dense-cored vesicles have a secretory function and contain precursors of elastin.Elongated vesicles or profiles containing collagen fibrils were observed in smooth muscle cells and fibroblasts. In the cell periphery, these vesicles were often found to communicate with the extracellular space. Further inside the cells, they showed a close spatial relationship to the Golgi complex. Neither thiamine pyrophosphatase nor acid phosphatase activity was demonstrated in the elongated vesicles. Like the plasma membrane, their limiting membrane was positively stained for alkaline phosphatase. On the basis of these findings and the absence of uptake of exogenous markers in them, it is suggested that the elongated vesicles represent a means for collagen secretion in the growing aortic wall. The Golgi complex is believed to be involved in the transfer of collagen to these vesicles.     

DIGESTION AND THE DISTRIBUTION OF ACID PHOSPHATASE IN BLEPHARISMA

Suspensions of Blepharisma intermedium were fed latex particles for 5 min and then were separated from the particles by filtration. Samples were fixed at intervals after separation and incubated to demonstrate acid phosphatase activity. They were subsequently embedded and sectioned for electron microscopy. During formation of the food vacuole, the vacuolar membrane is acid phosphatase-negative. Within 5 min, dumbbell-shaped acid phosphatase-positive bodies, possibly derived from the the acid phosphatase-positive Golgi apparatus, apparently fuse with the food vacuole and render it acid phosphatase-positive. A larger type of acid phosphatase-positive, vacuolated body may also fuse with the food vacuole at later stages. At about 20 min after formation, acid phosphatase-positive secondary pinocytotic vesicles pinch off from the food vacuoles and approach a separate system of membrane-bounded spaces. By 1 hr after formation, the food vacuole becomes acid phosphatase-negative, and the undigested latex particles are voided into the membrane-bounded spaces. The membrane-bounded spaces are closely associated with the food vacuole at all stages of digestion and are generally acid phosphatase-negative. Within the membrane-bounded spaces, dense, pleomorphic, granular bodies are found, in which are embedded mitochondria, paraglycogen granules, membrane-limited acid phosphatase-containing structures, and Golgi apparatuses. The granular bodies may serve as vehicles for the transport of organelles through the extensive, ramifying membrane-bounded spaces.