Two species of lysosomal organelles in cultured human fibroblasts.

Cultured diploid human skin fibroblasts were fractionated by a procedure that maximizes recovery of particles containing acid hydrolases. The cells were detached by controlled trypsinization, disrupted by N2 cavitation at low pressure and fractionated at 18,000 x g on a self-generating gradient of colloidal silica. This procedure separated two species of particles that could be consisered lysosomal. The denser one (peak density 1.11) was apparently free of other contaminants, but the more buoyant one (peak density 1.085) sedimented with or close to the peaks of other organelles, including mitochondria, Golgi, endoplasmic reticulum and plasma membranes. The two populations of particles contained acid hydrolases (phosphatase, six glycosidases and four cathepsins) in roughly equal proportions, displayed latency, had similar turnover of 35S-mucopolysaccharide in normal as well as in iduronidase-deficient cells, and were recipients of alpha-L-iduronidase, previously shown to be acquired by receptor-mediated endocytosis. Acid phosphatase staining of the intact fibroblasts showed residual bodies scattered throughout the cytoplasm and, near the nucleus, a prominent network of tubules and associated dilatations and knob-like enlargements. In both thin and thick sections, these appeared continuous, as if forming a three-dimensional network similar to the network described by Novikoff (1976) as GERL. Ultrastructural studies of the isolated fractions showed the denser lysosomal peak to be composed of small round or oblong acid phosphatase-positive bodies. The more buoyant peak contained the nonlysosomal organelles predicted from the biochemical markers, small acid phosphatase-positive bodies and large multivesiculated structures in which acid phosphatase was localized in a matrix surrounding apparently empty vesicles. These large structures may represent fragments of GERL. We suggest that the dense and buoyant lysosomal organelles originate primarily from residual bodies and the GERL network, respectively.     

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.     

Localization of acid phosphatase activity in collagen-secreting and collagen-resorbing fibroblasts

The ultrastructural localization of acid phosphatase (ACPase) activity was examined in cultured human gingival fibroblasts in the formative and resorptive phases. In the collagen-secreting fibroblasts, weak ACPase activity was demonstrated in the lysosomes, inner Golgi cisternae, and condensing vacuoles, and none was found in the Golgi-associated endoplasmic reticulum-lysosome system (GERL), presecretory granules, or secretory granules. On the contrary, collagen phagocytosis induced strong ACPase activity in the GERL, which was in addition to the weaker activity found in the same sites as those in the collagen-secreting cells. At the same time, collagen secretion was suppressed, and dense elongated secretory bodies associated with ACPase activity accumulated within the cells. When collagen fibrils had been interiorized in whole or in part within the phagosomes, primary lysosomes derived from the Golgi-GERL complex then fused with them to form phagolysosomes. Collagen degradation occurred within these bodies. The observations indicate significant differences in ACPase activity used as a marker for lysosomal enzyme activities in the different functional phases of fibroblasts. These results suggest that fibroblasts work only one way at a given time, viz., collagen synthesis or collagen degradation.     

Localization of acid phosphatase activity in collagen-secreting and collagen-resorbing fibroblasts

Summary The ultrastructural localization of acid phosphatase (ACPase) activity was examined in cultured human gingival fibroblasts in the formative and resorptive phases.In the collagen-secreting fibroblasts, weak ACPase activity was demonstrated in the lysosomes, inner Golgi cisternae, and condensing vacuoles, and none was found in the Golgi-associated endoplasmic reticulum-lysosome system (GERL), presecretory granules, or secretory granules. On the contrary, collagen phagocytosis induced strong ACPase activity in the GERL, which was in addition to the weaker activity found in the same sites as those in the collagen-secreting cells. At the same time, collagen secretion was suppressed, and dense elongated secretory bodies associated with ACPase activity accumulated within the cells. When collagen fibrils had been interiorized in whole or in part within the phagosomes, primary lysosome derived from the Golgi-GERL complex then fused with them to form phagolysosomes. Collagen degradation occurred within these bodies. the observations indicate significant differences in ACPase activity used as a marker for lysosomal enzyme activities in the different functional phases of fibroblasts.These results suggest that fibroblasts work only one way at a given time, viz., collagen synthesis or collagen degradation.     

Light- and electron-microscopic observations of the spinal ganglion cells of mice

Under the light microscope, large and small types of spinal ganglion cells can be seen in the mouse. Among those of the small type bipolar cells are observed. By electron microscopy, the cells of the large type have a low electron density and those of the small type a high electron density. Bipolar cells continue to be observed among the latter. The cells of the spinal ganglia contain different cellular apparatus in enormous quantity. There are four kinds of lysosomes in the GERL, i.e. the coated vesicles, dense bodies, multivesicular bodies and autophagic vacuoles.     

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.     

Fine structural and histochemical studies on salivary glands of Peripatoides novae-zealandiae (Onychophora) with special reference to acid phosphatase distribution

The Onychophora feed on small arthropods and produce saliva when ingesting prey. Although saliva undoubtedly helps to liquefy the food its constituents have not yet been fully described. The salivary glands, two long tubes of glandular epithelium, are known to secrete a powerful protease, however, besides other enzymes and mucus. In Peripatoides novae-zealandiae there are protein-secreting cells of three types, referred to here as columnar, cuboidal and modified cells, and mucus cells. The anterior two-thirds of the gland show most cell diversity, while the posterior region consists mainly of columnar cells. These are the most numerous elements overall and they probably secrete salivary protease. In thick resin sections the granules of all protein-secreting cells stain strongly with methylene blue. Those of columnar cells are markedly uneven in size and accumulate distally, eventually filling the cytoplasm. More proximal Golgi regions may be discernible. Mucus cells are all of one type and their secretion droplets are stained lightly by methylene blue. The electron microscope shows that distal microvilli, desmosomes and septate junctions are common to all gland cells. In columnar cells, secretory material is contributed by Golgi complexes and by rough endoplasmic reticulum. Early secretory vacuoles containing dense material are seen in the concavity of Golgi regions. They are precursors to larger condensing vacuoles whose contents have a more flocculent appearance, and which may attain 3–4 μm in diameter. These evolve into secretory granules, usually of uneven texture, which are up to 2–5 μm in diameter. Histochemical tests for acid phosphatase show moderate amounts of enzyme throughout the gland. In whole mounts and sections the strongest reaction is in a band of cuboidal cells along the anterior median border. Columnar cells show a diffuse cytoplasmic reaction towards the base and sometimes distal to the nucleus, and mucus cells may also react strongly round the nucleus. Cytoplasm near the lumen shows little reaction. The secretory granules do not appear to contain active enzyme. Under the electron microscope a positive reaction for acid phosphatase is seen in lysosomal derivatives near the base and lateral periphery of gland cells. These bodies are probably autophagic vacuoles and they may contain membranous whorls and possibly old secretion granules. Acid phosphatase is involved also in the elaboration of new secretory granules in both columnar and mucus cells. Dense reaction product is found in a system of interconnected tubules and cisternae near the innermost face of the Golgi complex, which is interpreted as GERL. Acid phosphatase is present in the peripheral zone of adjacent early secretory vacuoles, and interconnections occur between GERL and secretory vacuoles. It is suggested that GERL tubules containing the enzyme may fuse with early secretory vacuoles and release acid phosphatase at their periphery. The acid phosphatase reaction is negative in large condensing vacuoles and most secretory granules. These findings are consistent with what is known from mammalian cells, including those of salivary glands.     

豚鼠Clara细胞GERL及其在低氧下变化的细胞化学观察

本文对正常和模拟海拔5000米高度24、48和72小时后豚鼠Clara细胞GERL进行了超微结构和ACP活性的电镜酶细胞化学观察。结果表明Clara细胞具发达的GERL,包括ACP阳性的致密膜囊、致密囊泡、有衣小泡和细胞衣被覆区,Clara细胞GERL参与了分泌颗粒的形成。在低氧下GERL肥大,ACP阳性分泌颗粒增多,提示低氧导致GERL功能亢进,形成分泌颗粒的作用增强。     

Ultrastructural study of GERL in beige mouse alveolar macrophages

Alveolar macrophages of the beige mouse mutant have a system of smooth- surfaced elements with the hallmarks of GERL. GERL also appears to produce residual bodies, and both organelles show cytochemically demonstrable acid phosphatase activity. When cells are exposed to colloidal silver, the tracer is endocytosed via pinocytic vacuoles to GERL.     

Sites of lipoprotein particles in normal rat hepatocytes

Very low density lipoprotein (VLDL) particles are packaged by the Golgi apparatus into vacuoles which move to the plasma membrane and empty the particles into the space of Disse, via exocytosis. Traditionally, all lipoprotein-containing cisternae and vacuoles are thought to be parts of this pathway. Observations reported here demonstrate that there is a second population of lipoprotein-containing cisternae and vacuoles. This population is part of GERL, an organelle we consider to be a specialized hydrolase-rich region of the endoplasmic reticulum (ER). To our knowledge, this is the first systematic study of GERL in normal rat hepatocytes.     

Studies on the secretory process in exocrine pancreas cells. II. C57 black and beige mice

Published electron microscopic and cytochemical studies (thiamine pyrophosphatase and acid phosphatase) on exocrine pancreas cells of guinea pig, hamster, rat and rabbit have demonstrated that the nascent secretory granules, or condensing vacuoles, are part of GERL. The studies reported here show this to be true of the mouse pancreatic exocrine cells as well, thus permitting comparison of this cell type in the C57 black mouse and its "beige" mutant. This is of considerable interest because GERL is very much enlarged in these cells of the beige mouse. Most of GERL consists of wide dilated portions filled with electron-opaque materials that appear to be packaged into huge residual body-type lysosomes ("anomalous granules"). Acid phosphatase activity is demonstrable not only in these portions of GERL, but also in the condensing vacuoles as in pancreatic acinar cells in the black mouse where these dilated lysosome-producing regions are not present.     

A cytochemical study of acid phosphatase,thiamine pyrophosphatase and horseradish peroxidase in the Golgi-GERL complex of hepatoma ascites cells

Data from studies of ascitic cells of Chang hepatoma have shown that acid phosphatase (ACPase) can be localized simultaneously within the trans portion of the Golgi apparatus and in tubules of the Golgi-endoplasmic reticulum-lysosome (GERL) system. Reaction products of thiamine pyrophosphatase (TPPase) were also present consistently within trans elements of the Golgi apparatus and within GERL tubules. These new findings indicate that a close physiological association may exist between the Golgi apparatus and GERL, a concept that is consistent with previous observations of fibroblasts. When horseradish peroxidase (PO) is injected intraperitoneally into ascites-bearing rats and the ascitic cells withdrawn at different time intervals, PO could be localized within vesicles and tubules in the GERL region but could not be detected within the Golgi apparatus. Bulk-phase endocytosis requires a long time and a high concentration of PO to occur. The presence of PO within GERL indicates that this organelle may play a role in transporting or processing of certain exogenous proteins.     

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 effects of ACTH on acid phosphatase activity in endosomes, GERL and lysosomes of cultured adrenal tumor cells

Cultured cells derived from a mouse adrenocortical tumor transplant are unspecialized in appearance, but produce basal levels of steroids and demonstrate a near-immediate steroidogenic response to ACTH. There is biochemical evidence that ACTH induces increases in the uptake of serum lipoproteins by these cells and that this material is hydrolyzed in lysosomes to free cholesterol, a precursor for steroid end products. To investigate morphologically the role of lysosomes in the steroidogenic activity of these cells, cultures were incubated for 4 h with and without ACTH, then processed for the ultrastructural localization of acid phosphatase (ACPase), a marker enzyme for lysosomes, and for GERL, the lysosome-forming subcompartment of the Golgi, and examined by TEM and HVEM. Steroid output was determined by a fluorometric technique. Unstimulated cells secreted basal levels of steroids. By TEM, large endosomes, some containing semi-compact material and ACPase reaction product, were occasionally seen at the cell periphery and in the Golgi region. The Golgi and GERL were poorly developed. Residual bodies, a few of them ACPase+, appeared in the Golgi region and in microtubule-associated clusters near the cell membrane. ACTH-stimulated cells secreted steroids at 8-10 fold basal values. In TEM records, they displayed numerous ACPase+ endosomes between the cell periphery and the Golgi. The Golgi and GERL regions appeared to be hypertrophied and many large, inclusion-containing, strongly ACPase+ residual bodies appeared here and in elongated microtubule-containing cell processes. HVEM micrographs showed more definitively that ACTH produced distinct increases in the size of GERL and in the number of ACPase+ organelles. Our results suggest that in unstimulated cells, endosomes, presumably containing media-derived material, gain lysosomal enzymes in or near GERL, are transformed to residual bodies as their contents are hydrolyzed, and are subsequently translocated by microtubules to the cell periphery for exocytosis. ACTH appears to intensify all of these effects. The "giant" lysosomes seen in stimulated cells may result from a fusion of smaller lysosomes. Their amorphous contents may reflect an inefficient hydrolysis of LDL to free cholesterol.     

Ultrastructural and cytochemical studies on the developing adhesive disc of Boston Ivy tendrils

Summary Ultrastructural observations of the immature adhesive disc from tendrils of Boston Ivy showed that the peripheral cells, which are the presumptive contact layer, contain vacuoles of varied sizes which are filled with electron-dense aggregates. In small vacuoles, these deposits were appressed to the tonoplast and fusion of these small vacuoles with the large vacuoles apparently occurs. Cells from the central zone were largely parenchymatous. The vacuoles of many of these parenchyma cells contained electron-dense spheres and hemispheres of material either appressed to the tonoplast or within the vacuole lumen. In these cells, the vacuole-cytoplasm interface was characterized by a filiform network of interconnected membranes. Positive reactions with reagents for the identification of polyphenols indicate that the vacuoles of nearly all the peripheral cells and scattered cells of the central zone contain tanniniferous substances. Insoluble carbohydrates also occur in the vacuoles of the peripheral cells, but they contain little or no protein or lipid.     

The corpus luteum of the guinea pig. II. Cytochemical studies on the Golgi complex, GERL, and lysosomes in luteal cells during maximal progesterone secretion

This study characterizes the cytochemical properties of the Golgi complex, the structure which corresponds to Golgi complex-endoplasmic reticulum-lysosomes (GERL), and the granule population in luteal cells of guinea pigs at the time of maximum progesterone secretion, in material fixed by vascular perfusion, a method particularly suited for preserving both fine structure and enzyme activity. The distribution of several marker enzymes was determined by electron microscope cytochemistry. Acid phosphatase (ACPase) and arylsulfatase were used to identify structures containing lysosomal proteins. To resolve specific problems, additional cytochemical markers were employed: localization of thiamine pyrophosphatase (TPPase) (in the Golgi complex) and alkaline phosphatase (ALPase) (a plasma membrane marker), and prolonged osmication (a generally accepted method of marking the outer cisterna of the Golgi complex). The results demonstrate that at the time of peak steroid secretion the Golgi complex in luteal cells, in marked contrast to that of most other cell types, typically displays intense ACPase activity in all of its cisternae. Similarly, all Golgi cisternae stain after prolonged osmication and may show TPPase activity. On the other hand, GERL in luteal cells of this age, unlike that in most cells, commonly shows low levels of, or lacks, ACPase activity. However, GERL resembles that of other cell types in being TPPase-negative and in being unstained by treatment with aqueous OsO4. GERL and some Golgi cisternae are reactive for ALPase. The granule population in luteal cells of this stage consists of lysosomes, multivesicular bodies, electrontransparent vacuoles, and microperoxisome-like bodies. These results form a base line with which luteolytic changes described in the companion study (Paavola, L.G. 1978. 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. J. Cell. Biol. 79:59--73.) can be compared.     

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.     

Effect of colchicine on the Golgi complex of rat pancreatic acinar cells

Colchicine administered to adult rats at a dosage of 0.5 mg/100 g of body weight effected a disorganization of the Golgi apparatus in pancreatic acinar cells. The results obtained after various periods of treatment (10 min to 6 h) showed (a) changes in all components of the Golgi complex, and (b) occurrence of large vacuoles that predominated in cytoplasmic areas outside the Golgi region. The alterations in Golgi stacks concerned elements of the proximal and distal side: (a) accumulation of transport vesicles, (b) formation of small, polymorphic secretion granules, and (c) alterations in the cytochemical localization of enzymes and reaction product after osmification. Transport vesicles accumulated and accompanied short, dilated cisternae, which lack mostly the reaction products of thiamine pyrophosphatase, inosine diphosphatase, and acid phosphatase, and osmium deposits after prolonged osmification. After 4 to 6 h of treatment, accumulated transport vesicles occupied extensive cellular areas; stacked cisternae were not demonstrable in these regions. The changes on the distal Golgi side included GERL elements: condensing vacuoles were diminished; they were substituted by small, polymorphic zymogen granules, which appeared to be formed by distal Golgi cisternae and by rigid lamellae. Unusually extended coated regions covered condensing vacuoles, rigid lamellae, and polymorphic secretion granules. A cytochemical distinction between Golgi components and GERL was possible neither in controls nor after colchicine treatment. The cytochemical alterations in Golgi components were demonstrable 20-30 min following administration of colchicine; at 45 min, initial morphological changes--augmentation of transport vesicles and formation of polymorphic zymogen granules--became apparent. 20 min after administration of colchicine, conspicuous groups of large vacuoles occurred. They were located mostly in distinct fields between cisternae of the endoplasmic reticulum, and were accompanied by small osmium--reactive vesicles. Stacked cisternae were not demonstrable in these fields. Vacuoles and vesicles were devoid of reaction products of thiamine pyrophosphatase, inosine diphosphatase, and acid phosphatase. The results provide evidence that formation of stacked Golgi cisternae is impaired after colchicine treatment. The colchicine--induced disintegration of the Golgi complex suggests a regulatory function of microtubules in the organization of the Golgi apparatus.     

Ultrastructure of skin fibroblasts in storage diseases (mucopolysaccharidosis types IV and VI)

Ultrastructure of skin fibroblasts was studied in patients with mucopolysaccharidoses of types IV and VI and their relatives. In MPS VI and keratan-nonexcreting form of MPS IV the cytoplasm of fibroblasts contained numerous vacuoles with material of various electron density. The ultrastructure of cell did not differ from the norm in the keratan-excreting form of MPS IV. Skin fibroblasts from parents and siblings with MPS VI were found to contain a large number of residue bodies. The possible usage of ultrastructural data for early diagnosis of MPS and in medical genetic consultation of families of the patients with MPS is discussed.     

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