Cytochemical analysis of organelle degradation in phagosomes and apoptotic cells of the mucoid epithelium of mice.

The activity of mitochondrial cytochrome oxidase and peroxisomal catalase in the phagolysosomes and apoptotic bodies of mucoid epithelial cells was analysed. Tissue from 2-6 day old mice was used. The activity of acid phosphatase in lysosomes was also estimated. Cytochrome oxidase was demonstrated in well-preserved mitochondria inside phagosomes. Mitochondria in cells exhibiting apoptotic death also show activity of cytochrome oxidase. The enzyme activity in swollen mitochondria ceases before the membranes of the cristae disappear completely. Apoptotic bodies are phagocytosed by sister mucoid cells and, later on, they are digested inside the cell. Phagosomes which contain already degraded mitochondria show still active catalase in sequestered peroxisomes. The acid phosphatase involved in degradation of phagocytosed material originates from endocytosed lysosomes and primary and secondary lysosomes which fuse with the membranes of phagosomes.     

Isolation of peroxisomes from the dog kidney cortex.

The present study was undertaken to separate peroxisomes of the dog kidney cortex by the methods of discontinuous sucrose density gradient and zonal centrifugation. The separation of subcellular particles was evaluated by measuring the activities of reference enzymes, beta-glycerophosphatase for lysosomes, succinate dehydrogenase for mitochondria, glucose-6-phosphatase for microsomes, and catalase and D-amino acid oxidase for peroxisomes. The activities of D-amino acid oxidase and catalase were mainly observed in fractions 1 and 2 (1.6 and 1.7 M sucrose) obtained by discontinuous sucrose density-gradient centrifugation. Small amounts of acid phosphatase and succinate dehydrogenase contaminated these fractions. Considerably higher activity of catalase was determined in the supernatant, while D-amino acid oxidase showed a lower activity. By the method of zonal centrifugation, the highest specific activities of catalase and D-amino acid oxidase were found in fraction 50 (1.73 M sucrose) with no succinate dehydrogenase, acid phosphatase or glucose-6-phosphatase activity. These results suggested that peroxisomes of dog kidney cortex were clearly separated in 1.73 M sucrose from mitochondria, lysosomes and microsomes by zonal centrifugation.     

Histochemical Evidence for the Presence of Lysosome-like Particles in root Meristem Cells of Vicia faba

The lysosomes of animal cells, as defined by biochemists, areparticles limited by a lipid-protein membrane and which containhydrolytic enzymes; they are inert until the permeability ofthe limiting membrane has been altered. In addition, the lysosomesin animal cells have been demonstrated also by histochemicalprocedures. Acid phosphatase may be used as a marker for lysosomes,and can be demonstrated by a modified Gomori procedure. Thus,in controlled-temperature frozen sections of plant tissue, ithas been possible to demonstrate granules which are inactivefor acid phosphatase until subjected to agents that will disruptlipid-protein structures, namely freezing and thawing, formaldehydeand heat. Whether such particles are the lysosomes is discussed.     

Cytochemical analysis of organelle degradation in phagosomes and apoptotic cells of the mucoid epithelium of mice

Summary The activity of mitochondrial cytochrome oxidase and peroxisomal catalase in the phagolysosomes and apoptotic bodies of mucoid epithelial cells was analysed. Tissue from 2–6 day old mice was used. The activity of acid phosphatase in lysosomes was also estimated. Cytochrome oxidase was demonstrated in well-preserved mitochondria inside phagosomes. Mitochondria in cells exhibiting apoptotic death also show activity of cytochrome oxidase. The enzyme activity in swollen mitochondria ceases before the membranes of the cristae disappear completely. Apoptotic bodies are phagocytosed by sister mucoid cells and, later on, they are digested inside the cell. Phagosomes which contain already degraded mitochondria show still active catalase in sequestered peroxisomes. The acid phosphatase involved in degradation of phagocytosed material originates from endocytosed lysosomes and primary and secondary lysosomes which fuse with the membranes of phagosomes.     

Effect of metabolic alterations on the density and the contents of cathepsins B, H and L of lysosomes in rat macrophages

Crude lysosomal preparations from non-cultured peritoneal rat macrophages were shown to separate into high-density fractions rich in cathepsin B and H and low-density fractions rich in cathepsin L when layered on Percoll density gradients. Morphologically, the heavy lysosome fractions were found to consist mainly of lysosomes labeled with gold particles for anti-(cathepsin B, H and L). The light lysosome fractions contained lysosomes labeled with anti-(cathepsin B, H and L) and many other contaminants. In addition, small vesicles labeled by anti-(cathepsin L) were detected in these fractions. Addition of calf serum to the cultured macrophages induced an increase in the density of lysosomes in both dose-dependent and time-dependent fashions. Cathepsins B, H and L all shifted to the heavy lysosome fractions following the addition of serum. Progressive increase in fluorescence-labeled calf IgG in the heavy lysosome fractions after its addition suggests that the continuous entrance of excess proteins to lysosomes causes an increase in their density. This idea is supported by the fact that the density of lysosomes increased in parallel with the accumulation of horseradish peroxidase taken up in the heavy lysosome fractions. Increase in the density of lysosomes after treatment with ethyl(2S,3S)-3[(S)-3-methyl-1-(3-methyl-butylcarbamoyl)]oxirane-2- carboxylate (E-64-d) was marked in the cells cultured with serum-containing medium but slight in serum-deprived cells. However, the level of pyruvate kinase, an autophagic sequestration marker in heavy autolysosomes from E-64-d-treated cells, was much higher in serum-deprived cells, indicating that the contribution of heterophagic sequestration towards an increase in the density of lysosomes is much greater than that of autophagy.     

Vector-mediated overexpression of catalase A in the yeast Saccharomyces cerevisiae induces inclusion body formation.

To study the morphological effects of overexpression of catalase A in yeast, the gene coding for catalase A was introduced into Saccharomyces cerevisiae on a multicopy vector. After induction of microbody biogenesis and catalase A expression by growth on oleic acid as sole carbon source, cells were analyzed by immunofluorescence and immunoelectron microscopy. In addition, overexpression of catalase A was studied by quantitative immunoblotting and by activity measurement. Quantitative immunoblotting resulted in a 16-fold difference between immunoreactive material from transformed and non-transformed cells. An 18-fold increase of enzyme activity was measured in transformed cells due to overexpression of catalase A from plasmid pAH521. Immunofluorescent staining of semithin sections of Lowicryl HM20-embedded cells with anti-catalase localized peroxisomes and--at a low percentage--larger particles. By immunoelectron microscopy, these larger structures could be identified as agranular, electron-dense aggregates which are morphologically clearly distinct from the cytoplasm and not bounded by a membrane. These structures, which have been named inclusion bodies, contain catalase A but not other peroxisomal enzymes like thiolase. These findings suggest that cells are capable of compensating for overproduced proteins by formation of particular types of structures.     

Gramicidin and ion transport in isolated liver mitochondria

1. Eight distinct acid-hydrolase activities present in cytoplasmic extracts from bone tissue occur in latent form to the extent of 50-70% of their total activity, depending on the enzyme. 2. This latency can be decreased or suppressed by exposure to Triton X-100 or to media of low osmotic pressure, by treatment in the Waring Blendor, and by freezing and thawing, but not by increasing the substrate concentration in the assay medium up to 10-fold the Michaelis constant of the enzymes. 3. Latency is the property of the particle-bound enzymes, and treatments that suppress latency simultaneously cause solubilization of the enzymes. Most enzymes show an excess of free over soluble activity; the magnitude of this excess seems to depend largely on the nature of the enzyme, and sometimes also on the kind of treatment suffered by the preparations; it is attributed mainly to adsorption artifacts. 4. In preparations subjected to graded activating treatments, seven of the eight acid hydrolases studied are released in closely parallel fashion, suggesting that they are associated with particles possessing similar properties. Acid phenylphosphatase is released less readily than the other enzymes by Triton X-100 and by exposure to media of low osmotic pressure. 5. It is concluded from these and previous published fractionation experiments that, with the possible exception of part of the acid-phenylphosphatase activity, the eight acid hydrolases studied belong to lysosome-like particles. Bone lysosomes exhibit a relatively high degree of biochemical and physical heterogeneity. Their possible functions are discussed. Part of the acid-phenylphosphatase activity could be linked to another group of particles. 6. Catalase is also partly (30%) latent in cytoplasmic extracts of bone. Latent catalase can be released by some of the treatments that suppress the latency of the lysosomal enzymes, but differs from the latter by a greater resistance to Triton X-100, and, especially, by a complete insensitivity to exposure to media of low osmotic pressure. It is concluded from these results that the catalase-containing particles are probably different from lysosomes, as they are in liver. 7. Cytochrome oxidase, which is presumably associated with the mitochondria, and alkaline phenylphosphatase, an enzyme occurring predominantly in the microsomal fraction, exhibited no latency under the conditions of the present experiments.     

The digestive cells of the hepatopancreas in Aplysia depilans (Mollusca, Opisthobranchia): ultrastructural and cytochemical study

Digestive cells are the most abundant cell type in the digestive diverticula of Aplysia depilans. These are tall columnar or club shaped cells, covered with microvilli on their apical surface. A large number of endocytic vesicles containing electron-dense substances can be found in the apical zone, but the presence of many heterolysosomes of large diameter is the main feature of these cells. Glycogen particles and some lipid droplets were also observed. Peroxisomes with a circular or oval profile were common, but crystalline nucleoids were not detected in them, although a dense spot in the matrix was observed in a few cases. These organelles were strongly stained after cytochemical detection of catalase activity. The Golgi stacks are formed by 4 or 5 cisternae, with dilated zones containing electron dense material. Arylsulphatase activity was detected in the Golgi stacks and also in lysosomes. Cells almost entirely occupied by a very large vacuole containing a residual dense mass seem to be digestive cells in advanced stages of maturation. The observation of semithin and ultrathin sections indicates that these very large vacuoles are the result of a fusion among the smaller lysosomes. Some images suggest that the content of these large vacuoles is extruded into the lumen of the digestive diverticula.     

The lysosomes of earthworm chloragocytes: biochemical and morphological characterization

Nine latent and sedimentable acid hydrolases have been detected in the homogenates of earthworm chloragocytes. Their full activity was revealed by treatment with Triton X-100, a Waring blender treatment, freezing and thawing, hypotonic media or incubation at pH 5 and 25 degrees C. Solubilization paralleled the activation of the enzymes. Together with kinetic studies, these results indicate that the acid hydrolases of the chloragocytes are inside typical lysosome-like particles whose membrane is impermeable to their substrates. It could be shown by density equilibration centrifugation that the lysosomes of those cells constitute a heterogeneous population of subcellular particles distinct from the chloragosomes. Moreover, their digestive function has been directly demonstrated by the capture and degradation of serum albumin. The lysosomes of the chloragocytes have been clearly identified as polyvesicular bodies by electron microscopic analysis of the fractions obtained by density equilibration centrifugation and by examination of the whole tissue, as such or after endocytosis of serum albumin or ferritin. Finally, our results do not support a possible relationship between the lysosomes and the chloragosomes of the chloragocytes.     

The significance of paravacuolar granules of the thyroid. A histologic, cytologic and ultrastructural study

The presence of the so-called "paravacuolar granules" in thyroid follicular cells has been associated with increased metabolic activity of the gland, regressive changes, degeneration, phagocytic activity and benign papillary hyperplasia. During the course of a review of the intraoperative cytologic preparations and corresponding histologic sections from 73 thyroid cases, the presence of granules within follicular cells was noted in 25 cases (18 adenomatous or colloid goiters, 3 follicular adenomas, 2 papillary carcinomas, 1 follicular carcinoma and in thyroid tissue surrounding a follicular adenoma in 1 case). Histochemical and ultrastructural studies showed the granules to consist of lysosomes containing hemosiderin or lipofuscin pigments. These findings indicate that the presence of paravacuolar granules in thyroid cells is a common nonspecific finding that simply reflects: (1) the erythrophagocytic capability of the follicular epithelial cells, which results in the accumulation of iron within lysosomes, and (2) the accumulation of lipofuscin pigments within lysosomes as a result of degradation of endogenous cellular material.     

Regulating secretory lysosomes

Secretory lysosomes are lysosomes which are capable of undergoing regulated secretion in response to external stimuli. Many cells of the immune system use secretory lysosomes to release proteins involved in their specialised effector mechanisms. Precisely how lysosomal secretion is regulated in each of these cell types is now the study of much research as these mechanisms control the ability of each of these cells to function. Studies on a number of human genetic diseases have identified some key proteins in controlling secretory lysosome release, and now many interacting partners have been identified. The different regulatory components seem to vary from one cell type to another, providing a multitude of ways for fine tuning the release of secretory lysosomes.     

Cytochemical demonstration of latency of lysosomal hydrolases in digestive cells of the common mussel,Mytilus edulis,and changes induced by thermal stress

Latent beta-glucuronidase and glucosaminidase activities have been demonstrated in small cytoplasmic particles, which may possibly be primary lysosomes, as well as some larger granules of the digestive cells of the common mussel. Latency was indicated by increased staining of these structures following incubation in buffer at pH 4.5 at 37 degrees C. The exposure of mussels to temperatures of 25-28 degrees C over a period of four days induced a significant decrease in the latency of lysosomal glucosaminidase. Thermal death produced labilization of lysosomes although selective release of hydrolase activity was indicated by the differential latency of glucosaminidase and glucuronidase. The injection of hydrocortisone induced a significant increase in latency in stressed animals, indicating that the stress response involved changes in structure and function of membranes.     

ELECTRON MICROSCOPY OF LYSOSOME-RICH FRACTIONS FROM RAT LIVER

A preliminary electron microscope study has revealed the presence in lysosome-rich fractions, isolated from rat liver, of hitherto undescribed cytoplasmic particles, called "dense bodies." Approximately 0.37 µ in length, the dense bodies often possess an internal cavity and external membrane. They contain many electron-dense granules 55 to 77 A, or less, in diameter. Such dense bodies are also visible in electron micrographs of parenchymatous cells in liver sections. The correlations between dense bodies and lysosomes are listed, but until pure preparations are available it is not possible to assert that dense bodies and lysosomes are identical.     

Comparison of the Kinetics of Maturation of Phagosomes Containing Apoptotic Cells and IgG-Opsonized Particles

Defective clearance of apoptotic cells has emerged as an important contributing factor to the pathogenesis of many diseases. Although many efforts have been made to understand the machinery involved in the recognition between phagocytes and potential targets, little is known about the intracellular transport of phagosomes containing apoptotic cells within mammalian cells. We have, therefore, performed a detailed study on the maturation of phagosomes containing apoptotic cells in a non-professional phagocytic cell line. This process was compared with the maturation of IgG-opsonized particles, which are internalized via the Fcγ-receptor (Fcγ-R), one of the best characterized phagocytic receptor, in the same cell line stably expressing the Fcγ-RIIA. By comparing markers from different stages of phagosome maturation, we have found that phagosomes carrying apoptotic particles reach the lysosomes with a delay compared to those containing IgG-opsonized particles. Enrichment of the apoptotic particles in phosphatidylserine (PS) neither changed the kinetics of their engulfment nor the maturation process of the phagosome.     

Receptor-mediated endocytosis of immunoglobulin-coated colloidal gold particles in cultured mouse peritoneal macrophages. Chloroquine and monensin inhibit transfer of the ligand from endocytic vesicles to lysosomes

Earlier studies have shown that immunoglobulin G (IgG)-coated colloidal gold particles bind to specific receptors on the macrophage surface and accumulate in coated pits. They are then internalized via endocytic vesicles and transferred to lysosomes. During this process the plasma membrane is depleted of binding sites for IgG, suggesting that both the receptor and the ligand end up in lysosomes. Here, we have examined the effects of the weak base chloroquine and the Na+-H+ ionophore monensin on endocytosis and intracellular transport of IgG-coated colloidal gold particles in cultured macrophages. The results indicate that chloroquine and monensin do not arrest uptake of IgG-coated particles bound to the cell surface. On the other hand, the drugs strongly inhibit transfer of the particles from endocytic vesicles to lysosomes, the latter marked by prior pulse-chase labeling of the cells with horseradish peroxidase. Since the main effect shared by chloroquine and monensin is to raise pH in acid compartments such as endocytic vesicles and lysosomes, the findings suggest that the transfer of IgG-coated particles into the lysosomes is a pH-dependent process. It remains to be shown whether it is the membrane fusion as such that is controlled by pH or, more specifically, the transfer of receptor-bound ligands into the lysosomes.     

The Microelectrophoretic Behaviour of Plant Mitochondria compared with Rat Mitochondria

‘Heavy’ mitochondrial preparations of bean, cauliflower,and rat liver have been found to give unimodal distributionfor electrophoretic mobility against number of particles. ThepH-mean mobility curves were similar in form and consistentwith the mitochondrial surfaces being lipoproteins. de Duve (1959) separated ‘heavy’ and ‘light’(lysosome-rich) mitochondrial fractions from rat liver. Microelectrophoreticstudies on similar ‘heavy’ and ‘light’mitochondrial preparations from rat liver have shown the latterto consist mostly of mitochondria with some faster-moving particlestentatively identified with de Duve's lysosomes. ‘Light’mitochondrial preparations of bean showed no evidence of particlesadditional to mitochondria.     

A ZONAL ROTOR METHOD FOR THE PREPARATION OF MICROPEROXISOMES FROM EPITHELIAL CELLS OF GUINEA PIG SMALL INTESTINE

A method is described for the preparation of catalase particles from homogenates made from suspensions of epithelial cells of the small intestine of the guinea pig. Electron microscope examination of the preparations revealed the presence of small diaminobenzidine-positive particles measuring 0.1–0.3 nm in diameter and resembling the microperoxisomes observed by Novikoff and Novikoff (1972. J. Cell Biol. 53:532.). Analytical data upon which the method is based are presented. The technique consisted of a rate zonal separation of microperoxisomes from large particles followed by an isopycnic separation from less dense organelles. Application of the method yielded microperoxisomes purified between 20- and 30-fold.     

Lysosomal nature of plant vacuoles I. Apparent absence of lysosomal particles in tomato fruit and leaf homogenates

Differential centrifugation of the subcellular particles oftomato fruit was carried out after brief homogenization of thetomato fruit with a blendor in 0.5 M sucrose, containing 0.2M Tris-HCl buffer, pH 8.0. About 60% of the catalase activitywas detected in the peroxisomal fraction. However, most of theacid phosphatase and carboxypeptidase, which are marker enzymesfor lysosomes, were detected in the soluble fraction, as wasglucose-6-phosphate dehydrogenase, a marker enzyme for solublecytosol. Similar results were obtained from differential centrifugationof the tomato leaf homogenate. This absence of the lysosomalparticles in the tomato fruit and leaves is discussed, particularlyas an indication of the presence of hydrolytic enzymes in thecentral vacuoles of mature plant cells. (Received February 3, 1975; )     

Acidification of endocytic compartments and the intracellular pathways of ligands and receptors

Several hormones, serum proteins, toxins, and viruses are brought into the cell by receptor-mediated endocytosis. Initially, many of these molecules and particles are internalized into a common endocytic compartment via the clathrin-coated pit pathway. Subsequently, the ligands and receptors are routed to several destinations, including lysosomes, the cytosol, or the plasma membrane. We have examined the mechanism by which sorting of internalized molecules occurs. A key step in the process is the rapid acidification of endocytic vesicles to a pH of 5.0-5.5 This acidification allows dissociation of several ligands from their receptors, the release of iron from transferrin, and the penetration of diphtheria toxin and some viral nucleocapsids into the cytoplasm. Transferrin, a ligand that cycles through the cell with its receptor, has been used as a marker for the recycling receptor pathway. We have found that in Chinese hamster ovary (CHO) cells transferrin is rapidly segregated from other ligands and is routed to a complex of small vesicles and/or tubules near the Golgi apparatus. The pH of the transferrin-containing compartment is approximately 6.4, indicating that it is not in continuity with the more acidic endocytic vesicles which contain ligands destined to be degraded in lysosomes.