Distribution of Acid Phosphatase in Paramecium caudatum: Its Relations with the Process of Digestion

SYNOPSIS. The distribution of acid phosphatase was investigated at the ultrastructural level in Paramecium caudatum. Acid phosphatase occurs in endoplasmic reticulum, Golgi apparatus, food vacuoles, autophagic vesicles, vacuolar and dense bodies. Some slight deposits are also seen in the mitochondria.
These observations point out that this hydrolase activity is related to digestive processes. The enzyme, originating from the endoplasmic reticulum and Golgi apparatus reaches the food vacuole or autophagic vesicle likely via the reticulum. The digestion of the bacteria or of the enclosed organelle gives rise to electronopaque material which is later found in dense bodies. These dense bodies are likely secondary lysosomes and it is possible that they may fuse with the young food vacuole or with autophagic vesicles.     

大鼠睾丸间质细胞的自体吞噬活动

本文结合超微结构和细胞化学观察,研究大鼠睾丸间质细胞(Leydig细胞)中溶酶体的结??构与功能。观察结果表明,大鼠睾丸间质细胞中高尔基体非常发达,在高尔基体的成熟面存在着CMP酶阳性反应的GERL系统,说明这种细胞有不断产生溶酶体的能力。细胞化学结果也证实在睾丸间质细胞有较多的初级和次级溶酶体。睾丸间质细胞不仅有较多的溶酶体,而且还有相当数量的自噬小体,存在着活跃的自体吞噬活动。自噬小体的界膜来源于特化的光面内质网或高尔基体膜囊,包围的内容物主要是光面内质网和少量线粒体。当自噬小体与溶酶体融合后即成为自体吞噬泡,由于酶的消化作用,自体吞噬泡内的细胞器有一系列形态变化。根据CMP酶细胞化学反应,可以区分自噬小体和自体吞噬泡,后者是一种次级溶酶体,呈CMP酶阳性反应。睾丸间质细胞是分泌雄性激素的内分泌细胞,其光面内质网和线粒体在类固醇激素分泌中起重要作用,自体吞噬活动的结果是去除部分内质网和线粒体,可能在细胞水平上起着对雄性激素分泌的调节作用。     

LYSOSOMAL PACKAGING IN DIFFERENTIATING AND DEGENERATING ANURAN LATERAL MOTOR COLUMN NEURONS

The role of the Golgi apparatus and the Golgi-endoplasmic reticulum-lysosome complex (GERL) in the genesis of lysosomes was examined in differentiating and degenerating motor neurons of anuran larvae. Acid phosphatase, aryl sulfatase, and thiolacetic acid esterase were utilized as marker enzymes for the lysosomal system, while nucleoside diphosphatase and thiamine pyrophosphatase labeled the inner saccule(s) of the Golgi apparatus. Reduced osmium tetroxide was routinely deposited in the outer Golgi saccule regardless of the state of neuronal maturation. In all young neurons, the disposition of acid hydrolase reaction product paralleled the formation of GERL, with no lytic activity in the Golgi apparatus per se. Hypertrophy of the Golgi apparatus and GERL was observed in the early phases of degeneration, and both organelles apparently exhibit extensive hydrolytic activity. Dense bodies, autophagic vacuoles, and primary lysosomes were found arising from GERL, while the Golgi apparatus may produce primary lysosomal granules during regression. On the other hand, in differentiating neurons, hydrolytic activity was restricted to GERL and an occasional dense body and autophagic vacuole. These studies illustrate a parallelism between the development of GERL and genesis of primary and secondary lysosomes during neuronal cytodifferentiation, and implicate GERL and possibly the Golgi apparatus in lysosomal packaging in degenerating neurons.     

The corpus luteum of the guinea pig. III. Cytochemical studies on the Golgi complex and GERL during normal postpartum regression of luteal cells, emphasizing the origin of lysosomes and autophagic vacuoles

The postpartum involution of corpora lutea was examined by electron microscope cytochemistry of guinea pig ovaries previously fixed by vascular perfusion, a method which produces optimal preservation of steroid-secreting cells and yet maintains enzyme activity. The intracellular digestive apparatus was identified through the localization of two acid hydrolases, acid phosphatase (ACPase) and arylsulfatase. Other marker enzymes localized were thiamine pyrophosphatase (in Golgi cisternae) and alkaline phosphatase (along plasma membranes). Prolonged osmication was used to mark the outer Golgi cisterna. The results demonstrate that luteal cell regression is characterized by a striking increase in the number of lysosomes and the appearance of numerous, double-walled autophagic vacuoles. Both lysosomes and the space between the double walls of autophagic vacuoles exhibit ACPase and arylsulfatase activity. In contrast to earlier periods, just before and during regression, Golgi complex-endoplasmic reticulum-lysosomes (GERL) is markedly hypertrophied, displaying intense acid hydrolase activity. On the basis of various criteria, GERL is proposed to function in the formation of lysosomes and autophagic vacuoles. Lysosomes seem to develop from GERL as focal protuberances of varying size and shape, which detach from the parent structure. Double- walled autophagic vacuoles, often large and complex in structure, initially are produced as GERL cisternae envelop small areas of cytoplasm. Lytic enzymes, perhaps furnished by the engulfing membranes and trapped lysosomes, presumably bring about digestion of the contents of these vacuoles, producing first aggregate-type inclusions, then, as the contents are further degraded, myelin figure-filled residual bodies. ACPase activity occasionally appears within smooth endoplasmic reticulum tubules and cisternae in advanced regression, possibly suggesting that lytic enzymes utilize this membrane system as an access route to GERL. These data indicate that cellular autophagy is a prominent mechanism underlying luteal cell involution during normal postpartum degeneration of guinea pig corpora lutea. Furthermore they suggest that in regressing luteal cells GERL is responsible for packaging acid hydrolases into lytic bodies.     

Interrelationships between Golgi, GERL and synaptic vesicles in the nerve cells of insect and gastropod ganglia.

In addition to demonstrating synaptic vesicles, staining with the zinc-iodide-osmium tetroxide (ZIO) method reveals the presence of positively reacting GERL membranes in association with the Golgi complex and lysosomes in the nerve cell bodies within ganglia from the locust Schistocerca gregaria and the gastropod molluscs, Limnaea stagnalis and Helix aspersa. A positive response to ZIO occurs in certain Golgi vesicles and saccules, in GERL (Golgi-endoplasmic-reticulum-lysosomes), in multivesicular bodies as well as residual bodies and in small vesicles and cisternae of axonal smooth endoplasmic reticllum (ER). The interrelationships between these organelles are considered in view of the similarity of the ZIO localization to phosphatase-rich sites in the neuronal perikarya and with respect to the possibility that components of the synaptic vesicles are formed in the Golgi region of the cell and migrate via the axonal smooth ER to the synaptic regions.     

OSMIUM STAINING OF ENDOPLASMIC RETICULUM AND MITOCHONDRIA IN THE RAT ADRENAL CORTEX

The zona fasciculata of the rat adrenal cortex synthesizes and secretes glucocorticoids. As observed after aldehyde fixation, the cells in this zone contain an extensive endoplasmic reticulum (ER), a small Golgi apparatus, a moderate number of lipid droplets, and abundant mitochondria with tubulovesicular cristae. Numerous areas within the endoplasmic reticulum and mitochondrial cristae appear clear. In addition, a small percentage of mitochondria encompasses large, clear areas. After immersion of finely minced adrenal cortex in unbuffered 2% OsO₄ (40–48 hr at 40°C), deposits of osmium are seen within the Golgi apparatus, the entirety of the ER, and occasionally within mitochondria. In some mitochondria, the deposits are within cristae; in others, within vacuoles; in still others, in both cristae and vacuoles. These localizations correspond best to the clear areas found in aldehyde-fixed tissue. Osmium is not deposited in lipid droplets, in bar-containing inclusions, in mitochondrial matrix inclusions, or in the peripheral, outer mitochondrial spaces. Addition of zinc-iodide to OsO₄ increases the amount of Golgi apparatus and mitochondrial staining. Adrenocorticotropin (ACTH) does not affect the localization of deposits; hypophysectomy decreases mitochondrial staining. This study (a) emphasizes the necessity for electron microscopic confirmation of osmium localization when this technique is used as a Golgi apparatus stain; and (b) suggests that the ER-staining pattern may be consistent in cells actively synthesizing steroids or steroid-like compounds.     

GOLGI APPARATUS, GERL, AND LYSOSOMES OF NEURONS IN RAT DORSAL ROOT GANGLIA, STUDIED BY THICK SECTION AND THIN SECTION CYTOCHEMISTRY

New insights into the ultrastructure and phosphatase localizations of Golgi apparatus and GERL, and into the probable origin of lysosomes in the neurons of fetal dorsal root ganglia and the small neurons of adult ganglia have come from studying thick (0.5–1.0 µ) as well as thin (up to 500 A) sections by conventional electron microscopy. Tilting the thick specimens, by a goniometer stage, has helped to increase our understanding of the three-dimensional aspects of the Golgi apparatus and GERL. One Golgi element, situated at the inner aspect of the Golgi stack, displays thiamine pyrophosphatase and nucleoside diphosphatase activities. This element exhibits regular geometric arrays (hexagons) of interconnected tubules without evidence of a flattened portion (saccule or cisterna). In contrast, GERL shows acid phosphatase activity and possesses small cisternal portions and anastomosing tubules. Lysosomes appear to bud from GERL. Osmium deposits, following prolonged osmication, are found in the outer Golgi element. Serial 0.5-µ and thin sections of thiamine pyrophosphatase-incubated material demonstrate that, in the neurons studied, the Golgi apparatus is a continuous network coursing through the cytoplasm. Serial thick sections of acid phosphatase-incubated tissue suggest that GERL is also a continuous structure throughout the cytoplasm. Tubules of smooth endoplasmic reticulum, possibly part of GERL, extend into the polygonal compartments of the inner Golgi element. The possible physiological significance of a polygonal arrangement of a phosphatase-rich Golgi element in proximity to smooth ER is considered. A tentative diagram of the Golgi stack and associated endoplasmic reticulum in these neurons has been drawn.     

Fine structural localization of thiamine pyrophosphatase and acid phosphatase activities in the mouse pancreatic acinar cell

The fine structural localization of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) was examined in pancreatic acinar cells of fasting and fed mice. The results were not affected by these conditions. TPPase activity was positive in two and sometimes three cisternae of the inner Golgi lamellae as well as in the condensing vacuoles of the trans area, but negative in the rigid lamellae and small vesicles of the trans area. AcPase activity was demonstrated in two and sometimes three cisternae of inner Golgi lamellae, condensing vacuoles, rigid lamellae, lysosomes and smooth or coated vesicles in the trans area. The inner Golgi lamellae and the condensing vacuoles were positive for both enzyme activities. From these facts, the lysosome is considered to be formed not only in the GERL system but also through the rough endoplasmic reticulum-Golgi apparatus route. It is reasonable to consider that Novikoff's GERL is not independent from the Golgi apparatus but represents a part of this organelle.     

LYSOSOMES IN THE RAT SCIATIC NERVE FOLLOWING CRUSH

Peripheral nerves undergoing degeneration are favorable material for studying the types, origins, and functions of lysosomes. The following lysosomes are described: (a) Autophagic vacuoles in altered Schwann cells. Within these vacuoles the myelin and much of the axoplasm which it encloses in the normal nerve are degraded (Wallerian degeneration). The delimiting membranes of the vacuoles apparently form from myelin lamellae. Considered as possible sources of their acid phosphatase are Golgi vesicles (primary lysosomes), lysosomes of the dense body type, and the endoplasmic reticulum which lies close to the vacuoles. (b) Membranous bodies that accumulate focally in myelinated fibers in a zone extending 2 to 3 mm distal to the crush. These appear to arise from the endoplasmic reticulum in which demonstrable acid phosphatase activity increases markedly within 2 hours after the nerve is crushed. (c) Autophagic vacuoles in the axoplasm of fibers proximal to the crush. The breakdown of organelles within these vacuoles may have significance for the reorganization of the axoplasm preparatory to regeneration. (d) Phagocytic vacuoles of altered Schwann cells. As myelin degeneration begins, some axoplasm is exposed. This is apparently engulfed by the filopodia of the Schwann cells, and degraded within the phagocytic vacuoles thus formed. (e) Multivesicular bodies in the axoplasm of myelinated fibers. These are generally seen near the nodes of Ranvier.     

The role of the Golgi complex in the isolation and digestion of organelles

The origin of the membranes and lytic enzymes involved in autophagy has been studied in metamorphosing insect fat body.The Golgi complex has two functions in the organelle destruction which takes place when fat body cells change their activities. (1) It gives rise to envelopes which externalize organelles scheduled for destruction. Microbodies, mitochondria and rough endoplasmic reticulum are sequentially removed from the cytoplasm by investment in isolation membranes. During the isolating phase, isolation membranes have the same osmiophilia as the outer saccular and microvesicular components of the Golgi complex, they do not contain lytic enzymes and they are specific in their adhesion to organelles scheduled for destruction. (2) The Golgi complex gives rise to lytic enzymes. Primary lysosomes which contain acid phosphatase fuse with the isolation bodies formed from invested organelles to become autophagic vacuoles. During this lytic phase, acid phosphatase is present in the inner saccules and microvesicular components of the Golgi complex, in the primary lysosomes seen fusing with isolation bodies and in autophagic vacuoles.     

Fine structural localization of thiamine pyrophosphatase and acid phosphatase activities in the mouse pancreatic acinar cell

Summary The fine structural localization of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) was examined in pancreatic acinar cells of fasting and fed mice. The results were not affected by these conditions. TPPase activity was positive in two and sometimes three cisternae of the inner Golgi lamellae as well as in the condensing vacuoles of the trans area, but negative in the rigid lamellae and small vesicles of the trans area. AcPase activity was demonstrated in two and sometimes three cisternae of inner Golgi lamellae, condensing vacuoles, rigid lamellae, lysosomes and smooth or coated vesicles in the trans area. The inner Golgi lamellae and the condensing vacuoles were positive for both enzyme activities. From these facts, the lysosome is considered to be formed not only in the GERL system but also through the rough endoplasmic reticulum-Golgi apparatus route. It is reasonable to consider that Novikoff's GERL is not independent from the Golgi apparatus but represents a part of this organelle.This study was supported by a grant from the Japan Educational Ministry     

Osteoblastic and osteoclastic differentiation of mononuclear cells facing the resorbing surface of uncalcified cartilage in the tibia of embryonic chick

Summary The resorbing region of uncalcified cartilage in the tibia of embryonic chick was studied using ³H-proline autoradiography, histochemistry, and horseradish-peroxidase tracers.At the cartilage-bone marrow interface, two kinds of cells (A and B) were identified. Type-A cells were elongated, contacted the matrix of the uncalcified cartilage directly, and possessed extensive rough endoplasmic reticulum, one or two juxtanuclear Golgi apparatus and cell membranes exhibiting prominent alkaline phosphatase activity. Type-B cells were round to oval, mononucleate (occasionally binucleate), and contained abundant mitochondria, vacuoles and vesicles, well-developed Golgi apparatus, and lysosomes. The lysosomes and the majority of vacuoles and Golgi lamellae of these cells showed prominent acid phosphatase activity. Type-B cells accumulated more horseradish-peroxidase reaction product in their vacuoles and vesicles than type-A cells. Thick, banded collagen fibrils were occasionally found in the matrix of the resorbing surface. ³H-proline autoradiography revealed small numbers of grains at the cartilage-bone marrow interface.These findings suggest that type-A cells have osteoblastic and type-B cells osteoclastic properties and are precursor cells of osteoblasts and osteoclasts, respectively. The appearance of a mineral phase in the resorbing cartilage is probably important for the differentiation of these cells.     

Ultrastructural localization of phosphatases in the testes of the domestic fowl: Acid phosphatase and thiamine pyrophosphatase

Summary ACPase and TPPase activity has been examined in the germinal epithelium of the testes in the domestic fowl. ACPase activity in spermatogonia and spermatocytes was confined to the Golgi complex. In spermatids ACPase activity was seen in the endoplasmic reticulum and nuclear envelope in the phase I and especially in the phase II (the elongating phase). This activity gradually decreased during the next phase III, and had disappeared in the final phase IV. The membrane body showed ACPase reaction in the small peripheral vacuoles and cisternal structures surrounding large central vacuoles. ACPase was also present in vesicles surrounding the developing tail. Late spermatids showed an abundance of autophagic vacuoles which had a complex array of ACPase positive delimiting membranes. In Sertoli cells ACPase activity was predominant in the lysosomes. TPPase activity was seen in the cisternae of the Golgi complex in spermatogonia and spermatocytes. In spermatids activity was present in the endoplasmic reticulum during the phase II, but it is lost in later stages. The smaller vacuoles and cisternal structures in the membrane body also showed reaction products. According to the present results it is thought likely that the smaller vacuoles and cisternal structures of the membrane body are of endoplasmic reticulum origin. The autophagic vacuoles in spermatids and the lysosomes of Sertoli cells are considered responsible for the degradation of residual bodies cast off by spermatids.     

The relative thickness of intracellular membranes in epithelial cells of the ventral lobe of the rat prostate

Synopsis The relative thickness of intracellular membranes of epithelial cells in the ventral lobe of the rat prostrate was measured by a densitometric method. Glutaraldehyde perfusion followed by ruthenium tetroxide immersion fixation appeared to be the most suitable method for membrane thickness measurements. By thickness, the membranes could be roughly subdivided into three groups. The inner and outer membranes of the mitochondrion made up the thinnest membranes of the cell. The second group of membranes consisted of the membranes of the rough-surfaced endoplasmic reticulum and the Golgi apparatus, the different faces of the latter organelle, and the Golgi vesicles. The thickest group of membranes included those of the cell membrane, secretory granules, condensing vacuoles, lysosomes, autophagic vacuoles and multivesicular bodies. The differences in thickness of the membranes are probably due to the varying protein/lipid ratio, and the qualities and proportions of the different lipids in the membranes.     

Development of compound trichocysts in the ciliatePseudomicrothorax dubius

Summary During the logarithmic growth of the ciliatePseudomicrothorax dubius associations between mitochondria, rough endoplasmic reticulum and dictyosomes have been observed. The Golgi apparatus is very active and it is suggested that, as a consequence of cytotic activity, the contents of the Golgi vesicles become incorporated into large irregular vacuoles as globular material. The large vacuoles develop into trichocysts and the dictyosome derived globules consolidate to ultimately form the rod-like arms of the trichocysts of theMicrothoracidae.     

Ultrastructural Localization of Acid Phosphatase in Starved Tokophrya infusionum*

SYNOPSIS. Young organisms of Tokophrya infusionum starved for several hr, are best suited for a study of the fine structure of this organism including the distribution of its organelles. Acid phosphatase was localized by a combined electron microscopy and cytochemical approach using modified Gomori methods. The enzyme was found in small dense bodies, spheroid vesicles, missile-like bodies, rough-surfaced endoplasmic reticulum, residue and autophagic vacuoles. The small dense bodies are thought to be primary lysosomes since electron micrographs show a) a continuity between the membrane of the rough-surfaced endoplasmic reticulum and that of the dense bodies and b) a connection between the contents of both structures when the dense bodies form from the endoplasmic reticulum.     

Studies of the secretory process in the mammalian exocrine pancreas. I. The condensing vacuoles

Phosphatase cytochemistry was used to distinguish between the Golgi apparatus and GERL (considered as a specialized region of endoplasmic reticulum [ER] at the inner [trans] aspect of the Golgi stack) in pancreatic exocrine cells of guinea pig, rat, rabbit, and hamster. The trans element of the Golgi stack exhibits thiamine pyrophosphatase (TPPase) but no acid phosphatase (AcPase) activity. In contrast, GERL shows AcPase but no TPPase activity. The nascent secretory granules, or condensing vacuoles, are expanded cisternal portions of GERL. Continuities of condensing vacuoles with rough ER are suggested, and it is proposed that some secretory components may have direct access to the condensing vacuoles from ER. Connections of Golgi apparatus with GERL were not seen.     

Photoreceptor membrane breakdown in the spider Dinopis: Localisation of acid phosphatases

Summary The ultrastructural localisation of acid phosphatases (AcPhs) during the normal daily breakdown of rhabdomere membrane in Dinopis has been examined using -glycerophosphate and p-nitrophenyl phosphate as substrates. Results are related to the classification of organelles in the receptors given by Blest, Powell and Kao (1978). Weak and infrequent reactions are obtained in multivesicular bodies (mvbs) and multilamellar bodies (mlbs) derived from them. Residual bodies (rbs) begin to react strongly as they lyse. Source of AcPhs is endoplasmic reticulum which has barely differentiated towards the GERL configuration; it becomes reactive as it is incorporated into secondary lysosomes. GERL tubules, Y-bodies and vesicles respond erratically and weakly, and are also incorporated into rbs. No evidence was found for a significant participation of Golgi bodies in these processes, and acid phosphatase cytochemistry fails to reveal a topographical relationship between GERL in these cells and Golgi saccules. Coated vesicle clusters found in the predawn receptive segments are AcPh-negative; this implies that their previous identification as GERL-derived Nebenkerne carrying hydrolytic enzymes to newly-formed mvbs (Blest, Kao and Powell, 1978) is dubious. Isolation bodies and autophagic vacuoles enclosing other organelles in pathological receptors give strong reactions while adjacent secondary lysosomes derived from rhabdomere membrane and associated GERL give weak ones. It is concluded that rhabdomere-derived rb lysis is more tightly regulated than other autophagic processes, and it is suggested that a high degree of control is necessary in a receptor which may repeat the autophagy of a large mass of transductive membrane at least 60–100 times in the course of its working life.The authors thank Professor D.T. Anderson F.R.S. for the use of field facilities at the Crommelin Biological Field Station of Sydney University at Warrah, Pearl Beach, New South Wales throughout all these studies; Dr. Gary Griffiths (EMBO, Heidelberg) and Dr. Alex Pyliotis (Biochemistry, SGS, Australian National University) for some helpful comments on acid phosphatase histochemistry; Sally J. Stowe for help in the field; and Rod Whitty and the staff on the Electron Microscopy Unit for advice and support. Figure 28 was prepared by Chris Snoek     

Elektronenmikroskopische Untersuchungen von Differenzierungsvorgängen bei Moosen

Summary In meristematic cells of the gemma of Riella helicophylla and in young bud cells from the protonema of Funaria hygrometrica the cell plate is formed by fusion of small vesicles originating from the Golgi apparatus. These spherical vesicles of about 0.1 m diameter have an electron dense centre, probably consisting of pectic substances or their precursors. The endoplasmic reticulum producing multivesicular bodies participate in cell plate formation too. Another cytoplasmic component forming the cell plate are coated vesicles, the origin of which is the Golgi apparatus and perhaps also the endoplasmic reticulum. In view of these observations the question of whether the endoplasmic reticulum or the Golgi apparatus forms the cell plate must be answered in this way: both endoplasmic reticulum and Golgi apparatus supply material for growth of the cell plate. Multivesicular bodies, coated vesicles and other small vesicles of unknown nature participate in the formation of the primary wall.

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Zum Teil finanziert mit Sondermitteln des Landes Niedersachsen an Prof. Dr. M. Bopp.     

EFFECTS OF ARGININE DEPRIVATION, ULTRAVIOLET RADIATION, AND X-RADIATION ON CULTURED KB CELLS : A Cytochemical and Ultrastructural Study

Cultured KB cells (derived from a human oral carcinoma) grown in monolayers were injured by one of three agents: starvation by arginine deprivation or treatment with high doses of either ultraviolet radiation or x-radiation. The different agents produced changes in nucleolar structure and varying accumulations of triglyceride and glycogen. All three agents produced an increase in number and size of lysosomes. These were studied in acid phosphatase preparations, viewed by both light and electron microscopy, and, occasionally, in vital dye, esterase, and aryl sulfatase preparations. Ultrastructurally, alterations in lysosomes suggested that "residual bodies" developed in a variety of ways, i.e., from the endoplasmic reticulum, multivesicular bodies, or autophagic vacuoles. Following all three agents the endoplasmic reticulum assumed the form of "rough" or "smooth" whorls, and, after two of the agents, arginine deprivation or ultraviolet radiation, it acquired cytochemically demonstrable acid phosphatase activity. Near connections between the endoplasmic reticulum and lysosomes raise the possibility that in KB cells, at least when injured, the endoplasmic reticulum is involved in the formation of lysosomes and the transport of acid phosphatase to them.