Autophagic-lysosomal and mitochondrial sequestration of [14C]sucrose. Density gradient distribution of sequestered radioactivity

[14C]Sucrose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, was sequestered by sedimentable subcellular particles during incubation of the cells at 37 degrees C. The sedimentation characteristics of particle-associated [14C]sucrose were different from the lysosomal marker enzyme acid phosphatase, suggesting an involvement of organelles of greater size than the average lysosome. Isopycnic banding in isotonic metrizamide/sucrose density gradients resolved two major peaks of radioactivity: a light peak (1.08-1.10 g/ml) coinciding with lysosomal marker enzymes, and a dense peak (1.15 g/ml), coinciding with a mitochondrial marker enzyme. The dense peak was preferentially associated with large-size particles having the sedimentation properties of mitochondria, and it was resistant to the detergent digitonin at a concentration which extracted all of the radioactivity in the light peak. Similarly the autophagy inhibitor 3-methyladenine prevented accumulation of [14C]sucrose in the light peak, while the radioactivity in the dense peak was unaffected. We therefore tentatively conclude that the light peak represents autophagic sequestration of [14C]sucrose into lysosomes (and probably autophagosomes) while the dense peak represents a mitochondrial uptake unrelated to autophagy.     

Effects of insulin on cardiac lysosomes and protein degradation

Hearts perfused in the absence of added insulin had 1) accelerated rates of protein degradation, as assessed by release of phenylalanine and tyrosine; 2) increased rates of release of seven other amino acids; 3) decreased lysosomal latency and sedimentable lysosomal enzyme activity; 4) increased numbers of autophagic vacuoles in cardiac muscle cells; and 5) decreased activity of beta-N-acetylglucosaminidase in dense lysosomes (1.06-1.09 g/ml), as compared to hearts perfused in the presence of the hormone. After 3 h of perfusion in the absence of insulin, the changes that developed in protein degradation, lysosomal latency, and sedimentability, and in enzyme activity in dense lysosomes, were reversed by insulin addition during 90 min of subsequent perfusion. These studies suggest a role for insulin in controlling the activity of the lysosomal system and the involvement of this system in protein degradation, particularly in insulin-deprived tissue.     

Effect of monensin on the neuronal ultrastructure and endocytic pathway of macromolecules in cultured brain neurons

1. The endocytic pathway of horseradish peroxidase (HRP) was investigated in the perikarya of cultured neurons by electron microscopy and enzyme cytochemistry. The tracer was observed in endocytic pits and vesicles, endosomes, multivesicular bodies, and lysosomes. It took approximate 15 min for the transfer of HRP from the exterior of the cell to the lysosomes. 2. Monensin induced distension of the Golgi apparatus and formation of intracellular vacuoles. When neurons were incubated with both monensin and HRP for 30 to 120 min, the number of HRP-labeled endosomes was greater than that in the monensin-free group, whereas the reverse was seen for HRP-positive lysosomes. The formation of HRP-positive lysosomes in monensin-treated cells was blocked by 47 to 79%. 3. These results indicate that the intracellular transport of the endocytosed macromolecule is pH dependent. It is also possible that the export of lysosomal enzymes is inhibited by monensin, resulting in an accumulation of the endosomes and a reduction of the lysosomes.     

Lysosomal storage causes cellular dysfunction in mucolipidosis II skin fibroblasts

Mucolipidosis II (ML-II) is a fatal inherited metabolic disease caused by deficiency of GlcNAc-phosphotransferase, which plays a role in generating the mannose 6-phosphate recognition marker on lysosomal enzymes. In ML-II, many lysosomal acid hydrolases are mistargeted out of cells, and lysosomes become filled with undigested substrates, which explains inclusion cell disease as an alternative name for this disease. In this study, we revealed various cellular phenotypes in ML-II skin fibroblasts. We quantitated phospholipid and cholesterol within cells and showed ~2-fold accumulation in ML-II as compared with normal cells. Lysosomal pH of ML-II cells was higher than that of normal cells (5.29 ± 0.08 versus 4.79 ± 0.10, p < 0.001). The proliferated lysosomes in ML-II cells were accumulated ~3-fold in amount as compared with normal cells. Intracellular logistics including endocytosis and mannose 6-phosphate receptor recycling were impaired in ML-II cells. To confirm whether these ML-II cellular phenotypes derive from deficient lysosomal acid hydrolases within lysosomes, we performed supplementation of lysosomal enzymes using a partially purified total enzyme mixture, which was derived from the conditioned culture medium of normal skin fibroblasts after NH(4)Cl treatment. This supplementation corrected all of the previously described ML-II phenotypes. In addition, the autophagic and mitochondrial impairment that we have previously reported improved, and inclusion bodies disappeared on electron micrography following total lysosomal enzyme supplementation. Our results indicate that various cellular phenotypes in ML-II are caused by the deficiency of many lysosomal enzymes and massive accumulation of undigested substrates.     

Lysosomes and tegument pathology in the chemotherapy of schistosomiasis with 1,7-bis(p-aminophenoxy)heptane (153C51).

One therapeutic oral dose (400 mg/kg) of 153C51 administered to infested mice caused pathological changes in the dorsal region of the tegument of male Schistosoma mansoni during the period 3--24 h after treatment. These changes occurred prior to the 'hepatic shift'. At the ultrastructural level they consisted of a gradual accumulation in the tegument epidermis of numerous membranous inclusions with the characteristics of residual lysosomes and changes in the localization of acid phosphatase, a lysosomal enzyme. It seemed likely that these changes were due to inhibition or exhaustion of enzyme in the epidermis, followed by re-synthesis of enzyme in the cell bodies and its export to the epidermis. The elimination of hydrolytic activity from lysosomes in the epidermis would explain the accumulation of residual lysosomes. Drug-treated parasites retained their disguise of host red blood cell ghost antigens as shown by indirect fluorescent antibody-labelling and, therefore, it seemed unlikely that immunological factors could be important in producing the tegument pathology.     

Characterization of the proteolytic compartment in rat hepatocyte nodules

Persistent liver nodules (hepatocyte nodules, neoplastic nodules) were produced in rat liver by intermittent feeding with 2-acetylaminofluorene. Dense bodies (secondary lysosomes) were purified and characterized from the nodules. The purity of the dense body fraction was 90%. The levels of various lysosomal enzyme activities were lower in these dense bodies in comparison with dense bodies from control liver. Similarly, protein degradation was 50% lower in dense bodies from liver nodules than in control liver. The number of autophagic vacuoles (AVs) in the nodular tissue increased considerably after 3 h vinblastine treatment. We have taken advantage of this expansion in an effort to isolate these organelles from liver nodules. Autophagic vacuoles have been isolated recently from liver and kidney but not from putatively premalignant liver nodules. Fraction purity of AVs from liver nodules was 95%. As with dense bodies, AVs from nodular tissue displayed lower activities of proteinases and lower rates of protein degradation when compared with their counterparts from normal liver tissue. Accordingly, the lower rate of overall protein degradation in liver nodules can be ascribed to a decrease in lysosomal activity. A diminished autophagic sequestration capacity is the most plausible explanation for the decreased rate of proteolysis in cells. This could conceivably give these nodular cells a growth advantage and assist in their selective outgrowth as well as in their transformation from neoplastic into true cancer cells.     

Biochemical and morphological characterization of primary kidney cell cultures from beige mutant mice

Summary Primary kidney cultures from adult beige-J (bg ^J/ bg ^J) mice were selected for epithelial cell growth using D-valine medium. After 2 weeks of attachment and proliferation in vitro, the cells form a confluent or nearly confluent monolayer that retains several phenotypic characteristics of the beige-J mutant. These include large, multilamellar inclusion bodies that are apparently dysmorphic lysosomes, and higher concentrations of neutral glycosphingolipids and dolichols than control cells. -Glucuronidase activity, used as a lysosomal enzyme marker, is not elevated in beige-J-cultured kidney cells compared with controls, as it is in the intact kidney. The high levels of -glucuronidase activity in both control and mutant cells may mask expression of this difference in vitro. The action of the beige-J mutation in kidney cells is thought to be due to a block in exocytosis that results in the accumulation of abnormal lysosomes and their components. The maintenance of the beige phenotype in vitro indicates that the mutation is not suppressed in primary kidney cell cultures. The expression of the beige phenotype in vitro should be useful for studies concerning the primary lesion of this mutation.     

Lysosomal heterogeneity of dipeptidyl peptidase II active on collagen-related peptides

The subcellular distribution of dipeptidyl peptidase II (DPP II) in the rat kidney cortex, as determined by subfractionation of the mitochondrial/lysosomal fraction by rate sedimentation, indicated that this enzyme is mainly associated with the large, fast sedimenting lysosomes (protein droplets). The small lysosomes, on the other hand, displayed considerable size heterogeneity as indicated by the broad distribution of DPP II; cathepsin B, and a tripeptidyl peptidase active on Gly-Pro-Met-2-naphthylamide at pH 4 (TPP 4). Cathepsin D and N-acetyl-beta-D-glucosaminidase were limited primarily to the slower-sedimenting, small lysosomes. Equilibrium banding in sucrose gradients of the two main DPP II-containing lysosomal populations showed that the large lysosomes banded at a density of 1.235-1.24 g/ml while small lysosomes banded at three densities: 1.11-1.15 g/ml (lysosomal fragments), 1.20 g/ml (light lysosomes), and 1.235 g/ml (dense lysosomes). Identical distribution pattern were obtained for DPP II using either Lys-Ala-7-(4-methyl)coumarylamide or Gly-Pro-2-naphthylamide as the substrate at pH 5.5 and 5.0, respectively. Notably, DPP II and TPP 4, and cathepsin B as well, gave banding densities and distributions that were consistent with a lysosomal localization. Since triplets of the Gly-Pro-X-type released by the TPP 4 are ideal substrates for DPP II, the integrated action of tripeptidyl and dipeptidyl peptidases could make a novel contribution to the renal depolymerization and reabsorption of polypeptides, in particular the proline-rich, collagen-derived sequences that possess repeating-triplet primary structures.     

Protein degradation in lysosomes from chemically induced malignant rat hepatoma

Hepatocellular carcinomas were induced in rat liver by exposing the animals to diethylnitrosamine and 2-acetylaminofluorene in combination with partial hepatectomy. Light and electron microscopy demonstrated that the general appearance of the tumour tissue was that of highly differentiated malignant hepatocytic cells. Morphometrically there was a difference between normal and malignant cells in that the entire lysosomal apparatus was twice as large in malignant cells as in normal cells. This was mainly due to an increase in the fractional volume of autophagic vacuoles. A total lysosomal fraction (dense bodies and autophagic vacuoles) was isolated and characterized from both control and tumour livers. Marker enzyme analysis showed that the lysosomal enzyme activities were significantly lower in malignant liver tissue. Injection of leupeptin, an inhibitor of cathepsins B, H, and L, into rats did not increase the fractional volume of autophagic vacuoles in tumour tissue as much as in normal liver tissue. The proteolytic rate was lower in the lysosomal fraction from hepatoma cell tissue compared with the lysosomal fraction from normal cell tissue. This could conceivably be due to the lower activities of lysosomal enzymes. However, if the recovery of lysosomes is taken into account no clear-cut difference in lysosomal proteolysis between control and malignant liver tissue was noted. Accordingly, in malignant liver tissue a proteolytic balance is obtained characterized by an increased fractional volume of AVs and lower rate of protein degradation in individual lysosomes.     

GSK-3-TSC axis governs lysosomal acidification through autophagy and endocytic pathways

Impaired lysosomal activity, which results in defective protein processing, waste accumulation, and protein aggregation, is implicated in a number of disease pathologies. Acidification of lysosomes is a crucial process required for lysosome function. Previously we showed that inhibition of glycogen synthase kinase-3 (GSK-3) enhanced lysosomal acidification in both normal and pathological conditions. However, how GSK-3 integrates into the lysosome networking is unknown. Here we show that inhibition of mTORC1 and increased autophagic activity are downstream to GSK-3 inhibition and contribute to lysosomal acidification. Strikingly, lysosomal acidification is also restored by GSK-3 inhibition in the absence of functional autophagy, and, independently of mTORC1. This is facilitated by increased endocytic traffic: We show that GSK-3 inhibition enhanced material internalization, increased recruitment of active Rab5 into endosomes, and increased Rab7/RILP clustering into lysosomes, all processes required for late endosome maturation. Consistently, in cells defective in endocytic traffic caused by either constitutively active Rab5, or, deletion of the Niemann-Pick C1 protein, GSK-3 inhibition could not restore lysosomal acidification. Finally we found that the tuberous sclerosis complex, TSC, is required for lysosomal acidification and is activated by GSK-3 inhibition. Thus, the GSK-3/TSC axis regulates lysosomal acidification via both the autophagic and endocytic pathways. Our study provides new insights into the therapeutic potential of GSK-3 inhibitors in treating pathological conditions associated with impaired cellular clearance.     

Presence of a lysosomal enzyme, arylsulfatase-A, in the prelysosome-endosome compartments of human cultured fibroblasts

Although endosomes and lysosomes are associated with different subcellular functions, we present evidence that a lysosomal enzyme, arylsulfatase-A, is present in prelysosomal vesicles which constitute part of the endosomal compartment. When human cultured fibroblasts were subfractionated with Percoll gradients, arylsulfatase-A activity was enriched in three subcellular fractions: dense lysosomes, light lysosomes, and light membranous vesicles. Pulsing the cells for 1 to 10 min with the fluid-phase endocytic marker, horseradish peroxidase, showed that endosomes enriched with the marker were distributed partly in the light lysosome fraction but mainly in the light membranous fraction. By pulsing the fibroblasts for 10 min with horseradish peroxidase conjugated to colloidal gold and then staining the light membranous and light lysosomal fractions for arylsulfatase-A activity with a specific cytochemical technique, the endocytic marker was detected under the electron microscope in the same vesicles as the lysosomal enzyme. The origin of the lysosomal enzyme in this endosomal compartment was shown not to be acquired through mannose 6-phosphate receptor-mediated endocytosis of enzymes previously secreted from the cell. Together with our recent finding that the light membranous fraction contains prelysosomes distinct from bona fide lysosomes and was highly enriched with newly synthesized arylsulfatase-A molecules, these results demonstrate that prelysosomes also constitute part of the endosomal compartment to which intracellular lysosomal enzymes are targeted.     

Accumulation of sialic acid in endocytic compartments interferes with the formation of mature lysosomes. Impaired proteolytic processing of cathepsin B in fibroblasts of patients with lysosomal sialic acid storage disease.

The impact of an altered endocytic environment on the biogenesis of lysosomes was studied in fibroblasts of patients suffering from sialic acid storage disease (SASD). This inherited disorder is characterized by the accumulation of acidic monosaccharides in lysosomal compartments and a concomitant decrease of their buoyant density. We demonstrate that C-terminal trimming of the lysosomal cysteine proteinase cathepsin B is inhibited in SASD fibroblasts. This late event in the biosynthesis of cathepsin B normally takes place in mature lysosomes, suggesting an impaired biogenesis of these organelles in SASD cells. When normal fibroblasts are loaded with sucrose, which inhibits transport from late endosomes to lysosomes, C-terminal cathepsin B processing is prevented to the same extent. Further characterization of the terminal endocytic compartments of SASD cells revealed properties usually associated with late endosomes/prelysosomes. In addition to a decreased buoyant density, SASD "lysosomes" show a reduced acidification capacity and appear smaller than their normal counterparts. We conclude that the accumulation of small non-diffusible compounds within endocytic compartments interferes with the formation of mature lysosomes and that the acidic environment of the latter organelles is a prerequisite for C-terminal processing of lysosomal hydrolases.     

Fine structural demonstration of acid phosphatase in rabbit germinal epithelium prior to induced ovulation

The germinal or surface epithelium covering rabbit Graafian follicles contains occasional small, dark, lysosome-like bodies. After an ovulatory dose of human chorionic gonadotropin (HCG) such bodies gradually increase in size and number. At 8 hr after HCG there is a maximal accumulation in the apical follicle cells; then the dense bodies decrease and just prior to ovulation, 9.5 hr after HCG, only few of them remain in the attenuated surface epithelium. Most of the growing membrane-surrounded bodies probably represent lysosomes, since electron microscopy combined with cytochemistry revealed that many of them contain the lysosomal "marker" enzyme, acid phosphatase. The role of sex steroids and prostaglandins regarding lysosomal growth and LABILization is discussed. The close temporal relation between disappearance of the apical surface epithelial lysosomes and disintegration of the underlying tunica albuginea gives further support to our working hypothesis that at least part of the "ovulatory enzymes" emanate from the surface epithelium.     

Endocytosis and chloroquine accumulation during the cell cycle of hepatoma cells in culture

Variations of endocytic and of lysosomal functions during the cell cycle have been investigated in synchronized hepatoma cells (derived from Morris hepatoma 7288c) by following the cellular uptake of horseradish peroxidase, dextran (mol wt. 70,000), and chloroquine. Cell fractionation and cytochemistry show that in asynchronously growing cells exposed for 1 h to 5 mg/ml peroxidase, the bulk of the enzyme taken up by the cells is found in phagosomes. By using the same experimental system with synchronized HTC cells, large variations of endocytosis are observed during the cell cycle. Peroxidase uptake is lowest during mitosis, increases 5--10 times during G1 phase, reaches a plateau, and finally decreases at the end of S phase and during G2 phase. A similar evolution is observed for the uptake of dextran (0.5 or 1 mg/ml), but it is likely that a significant part of the polysaccharide is still associated with the pericellular surface after 1 h. Moreover, dextran is transferred more slowly than peroxidase to lysosomes. Cellular accumulation of chloroquine is related to intralysosomal pH or to the buffering capacity of lysosomes. Our results show that this drug is taken up more rapidly during G1 and S phases while the rate of accumulation is lowest in mitotic cells. The results are discussed in relation to the modifications of the physical properties of lysosomes during the cell cycle observed previously by cell fractionation and electron microsocopy, and to the possible role of lysosomes in the initiation of mitosis. Cyclic changes of endocytosis in actively dividing cells are demonstrated by our observations and may induce large differences in the uptake rate of extracellular substances.     

Human parathyroid hormone (1-34) transiently increases the excretion of lysosomal enzymes into urine and the size of renal lysosomes.

It has been reported that the urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG), a lysosomal enzyme, transiently increases in human after treatment with human parathyroid hormone (hPTH)(1-34). We report here that hPTH(1-34) caused transient changes in the size and density of rat renal lysosomes following urinary excretion of NAG and other lysosomal enzymes tested. Percoll density gradient centrifugation revealed that hPTH(1-34) slightly but significantly increased the fraction of high density lysosomes (around 1.12 g/ml) 5-10 min after the treatment with hPTH(1-34), with a concomitant decrease in the fraction of intermediate density lysosomes (1.07-1.08 g/ml). On electron micrographs, some lysosomes in proximal tubules but not in distal tubules showed a change in morphology from circular to oval, and became enlarged and electron-dense 5-10 min after the treatment with hPTH(1-34). These responses to hPTH(1-34) were also reversible and transient. NAG excreted in urine after treatment with hPTH(1-34) had the molecular mass of a mature form in lysosomes and/or endosomes and was not a prepro-and/or pro-form of the enzyme. Thus, the changes in the density and size of renal lysosomes appear to be associated with the exocytosis of lysosomal enzymes by hPTH(1-34).     

Herpes simplex virus and human cytomegalovirus replication in WI-38 cells. III. Cytochemical localization of lysosomal enzymes in infected cells.

Cytochemical localization of the lysosomal enzymes acid phosphatase and arylsulfatase in cells infected by herpes simplex virus (HSV) or human cytomegalovirus (CMV) showed the following interactions between viruses and host cell lysosomes: (i) many enveloped progeny viruses were located within cytoplasmic vacuoles containing lysosomal enzyme activity; (ii) naked cytoplasmic capsids appeared to acquire an envelope by budding directly into lysosomes; and (iii) many of the cytoplasmic dense bodies that are characteristic of CMV-infected cells and are thought to represent noninfectious aggregates of CMV structural proteins (I. Sarov and I. Abady, Virology 66:464-473, 1975) also acquired a limiting membrane by budding into lysosomes. Autophagy of other cytoplasmic elements was not observed, suggesting that there is some specificity involved in the association of viral particles and CMV dense bodies with lysosomes. Despite the presence of potentially destructive hydrolases, there was little evidence of significant morphological damage to intralysosomal viruses, and high titers of infectious particles were released into the medium. It would therefore appear that significant levels of HSV and CMV infectivity normally persist even though many progeny particles are directly exposed to lysosomal enzymes.     

Internalization of β-adrenergic receptor binding sites: Involvements of lysosomal enzymes

Lysosomal enzymes were detected in a highly purified preparation of frog erythrocytes. Pretreatment of intact erythrocytes with lysosomotropic drugs reduced the number of soluble β-receptors in isoproterenol-treated cells, whereas the level of membrane-bound receptors in these cells was unaffected. Subcellular fractionation by Percoll gradient centrifugation revealed that one species of lysosomes (density: 1.15 g/ml), contained a fraction of membrane-bound β-adrenergic receptors. This fraction of membrane-bound receptors was markedly increased when desensitized cells were pretreated with chloroquine. Thus the internalized receptors appear to be delivered to lysosomes and released from the endocytic vesicles by the lysosomal enzymes.     

An unusual lysosome compartment involved in vitellogenin endocytosis by Xenopus oocytes

We have investigated the lysosomal compartment of Xenopus oocytes to determine the possible role of this organelle in the endocytic pathway of the yolk protein precursor, vitellogenin. Oocytes have lysosome-like organelles of unusual enzymatic composition at all stages of their development, and the amount of hydrolase activity increases steadily throughout oogenesis. These unusual lysosomes appear to be located primarily in a peripheral zone of oocyte cytoplasm. At least two distinct populations of lysosomal organelles can be identified after sucrose density gradient fractionation of vitellogenic oocytes. Most enzyme activity resides in a compartment of large size and high density that appears to be a subpopulation of yolk platelets that are less dense than most platelets within the cell. The appearance of this high density peak of lysosomal enzyme activity coincides with the time of onset of vitellogenin endocytosis during oocyte development. The data suggest that endocytic vesicles that contain vitellogenin fuse with modified lysosomes shortly after their internalization by the oocyte. Pulse-chase experiments with radiolabeled vitellogenin suggest that the ligand passes through the low density platelet compartment en route to the heavy platelets. The accumulation of yolk proteins apparently results from a failure of these molecules to undergo complete digestion after their entry into an unusual lysosomal compartment. The yolk platelets that these proteins finally enter for prolonged storage appear to be a postlysosomal organelle.     

Conversion of dense lysosomes into light lysosomes during hepatocytic autophagy

Incubation of isolated rat hepatocytes under conditions which support maximal autophagy (amino acid-free medium) caused a marked alteration in the density distribution of lysosomes in continuous metrizamide gradients (mean peak density reduced from 1.14 to 1.09 g/ml). The autophagic sequestration inhibitor 3-methyladenine (3MA) partially prevented the density shift, presumably by stopping the formation of light autophagosomes which otherwise fuse with dense lysosomes and thereby alter the lysosomal density.     

Stimulation of the D5 dopamine receptor acidifies the lysosomal pH of retinal pigmented epithelial cells and decreases accumulation of autofluorescent photoreceptor debris

Optimal neuronal activity requires that supporting cells provide both efficient nutrient delivery and waste disposal. The incomplete processing of engulfed waste by their lysosomes can lead to accumulation of residual material and compromise their support of neurons. As most degradative lysosomal enzymes function best at an acidic pH, lysosomal alkalinization can impede enzyme activity and increase lipofuscin accumulation. We hypothesize that treatment to reacidify compromised lysosomes can enhance degradation. Here, we demonstrate that degradation of ingested photoreceptor outer segments by retinal pigmented epithelial cells is increased by stimulation of D5 dopamine receptors. D1/D5 receptor agonists reacidified lysosomes in cells alkalinized by chloroquine or tamoxifen, with acidification dependent on protein kinase A. Knockdown with siRNA confirmed acidification was mediated by the D5 receptor. Exposure of cells to outer segments increased lipofuscin-like autofluorescence, but SKF 81297 reduced autofluorescence. Likewise, SKF 81297 increased the activity of lysosomal protease cathepsin D in situ. D5DR stimulation also acidified lysosomes of retinal pigmented epithelial cells from elderly ABCA4(-/-) mice, a model of recessive Stargardt's retinal degeneration. In conclusion, D5 receptor stimulation lowers compromised lysosomal pH, enhancing degradation. The reduced accumulation of lipofuscin-like autofluorescence implies the D5 receptor stimulation may enable cells to better support adjacent neurons.