Characterization of basal lysosomes in exocrine acinar cells

Exocrine acinar cells possess a unique system of basally located lysosomes. Cytochemically, these lysosomes do not contain acid phosphatase, but react positively for trimetaphosphatase (C Oliver: J Histochem Cytochem 28:78, 1980). The present study extends the morphological and cytochemical characterization of these lysosomes in pancreatic, parotid, and exorbital lacrimal acinar cells from Sprague-Dawley rats and National Institutes of Health Swiss mice. The basal lysosomes are highly pleomoric in nature, and frequently appear as a system of anastomosing tubules of varying width. The lysosomes have a close morphological relationship with both the rough endoplasmic reticulum and mitochondria. In addition to trimetaphosphatase activity, the lysosomes are reactive for aryl sulfatase B, thiolacetic acid esterase, and cholinesterase. Since the cholinesterase activity could not be inhibited by specific inhibitors, this activity is most likely due to the presence of nonspecific esterases. The results of this study confirm the lysosomal nature of the basal lysosomes and underscore the necessity of using multiple enzyme activities to identify and characterize lysosomes.     

Structural changes in the digestive lysosomal system of sentinel mussels as biomarkers of environmental stress in mussel-watch programmes

A field study has been carried out to validate the measure of structural changes in the digestive lysosomal system of sentinel mussels as biomarkers of environmental stress. Previous laboratory studies demonstrated that the digestive lysosomal system of molluscs reponds to a variety of pollutants and to different stress situations by exhibiting significant changes in its structure. Mussels were collected monthly over 1 year at the Abra estuary (Bizkaia, Biscay Bay) from six sites with different degrees of pollution. The changes in the structure of the digestive lysosomes were quantified on cryostat sections of the digestive gland by means of automated image analysis. Four stereological parameters were recorded: lysosomal volume density, surface density, surface-to-volume ratio and numerical density. A seasonal pattern in the structure of the digestive lysosomes was evidenced with reduced volume, surface, size and numbers of lysosomes in winter-spring; increased volume, surface, size and numbers in summer and an intermediate situation in autumn. The structure of digestive lysosomes was also dissimilar among sites, the most significant differences being found between Plentzia (nonpolluted site) and Galea (polluted site). The digestive lysosomes of mussels collected from Galea were smaller and more abundant than in Plentzia's mussels in most sampling times. The basis of these differences are discussed to conclude that organic chemical pollution might be the cause for these specific changes which are different from the enlargement of digestive lysosomes described as a result of various sources of environmental stress. It is concluded that structural changes in the digestive lysosomes of sentinel mussels are sensitive to pollution-induced environmental stress even in the complex situation of the field where many factors may interact to affect the structure of the digestive lysosomal system.     

Inhibition of weak-base amine-induced lysis of lysosomes by cytosol

Certain amines known to be concentrated in lysosomes, termed "lysosomotropic amines," cause the formation of lysosomal vacuoles. A cell-free system was established to examine the effects of basic substances and acidic ionophores. In this system, the drugs not only increased the internal pH, but also caused a disruption of lysosomes. The osmotic swelling of lysosomes induced by protonated bases or cations for particular ionophores, which had accumulated within lysosomes driven by the proton pump, caused the osmotic lysis of lysosomes. The lysosomal disruption was inhibited upon the addition of the cytosol fraction. This phenomenon provides an in vitro system for studying the osmo-regulation and intercellular dynamics of the lysosomal system, including membrane fusion. The lysosomal stabilization factor was purified from the cytosol fraction and identified as ATP-stimulated glucocorticoid receptor translocation promoter (ASTP).     

Biogenesis of lysosomes in marshall cells and in cells of the male reproductive system

The mechanism of plasma membrane trafficking and degradation is still poorly understood. This investigation deals with the biogenesis of lysosomes during endocytic flow in Marshall cells and in various cell types of the male reproductive system. Marshall cells were exposed to ammonium chloride (NH4Cl) and leupeptin after labeling with cationic ferritin. In some experiments, the treated cells were immunogold labeled with anti-prosaposin antibody. NH4Cl and leupeptin are lysosomotropic agents that affect the endosomal-lysosomal progression. Testes, efferent ducts and epididymis from mouse mutants with defects affecting plasma membrane degradation were also used to analyze this process. NH4Cl produced a retention of cationic ferritin in endosomes and hindered the endosomal/lysosomal progression. Leupeptin did not affect this process. NH4Cl decreased the labeling of prosaposin in endosomes and lysosomes, while leupeptin increased the labeling of prosaposin in lysosomes. The number of lysosomes per cytoplasmic area was higher in treated cells than in controls. These findings suggest that leupeptin affected lysosomes whereas NH4Cl affected both endosomes and lysosomes. The endosomal and lysosomal accumulation of prosaposin induced by the treatment with NH4Cl and leupeptin indicated that the site of entry of prosaposinwas both the lysosome and endosome. Electron microscopy (EM) of tissues from mouse mutants with defects affecting plasma membrane degradation substantiated these observations. The EM analysis revealed a selective accumulation of multivesicular bodies (MVBs) and the disappearance of lysosomes, in testicular fibroblasts, nonciliated cells of the efferent ducts and principal cells of the epididymis, suggesting that MVBs are precursors of lysosomes. In conclusion: (1) endosomes and MVBs are a required steps for degradation of membranes; (2) endosomes and MVBs are precursors of lysosomes; and (3) endosomes, MVBs, and lysosomes appear to be transient organelles.     

Fine structure of the lysosomes in the two types of synoviocytes of normal rat synovial membrane

Summary The lysosomal system of the two types of synoviocytes (A and S) from the knee joint of normal rat synovial membrane was studied by electron-microscopic acid phosphatase cytochemistry. In random sections of the synovial intima lysosomes were more often encountered in the A-cell profiles than in the S-cell profiles. Characteristically, type-A synoviocytes showed many large and medium-sized lysosomes the cytochemical appearance of which varied considerably. No acid phosphatase activity was detectable in the cisternae of the Golgi apparatus or in the Golgi vesicles. In type-S synoviocytes the lysosomes were smaller, and more uniform in cytochemical appearance. Heavy deposits of acid phosphatase reaction product were constantly demonstrated in cisternae of the Golgi apparatus as well as in smooth-walled Golgi vesicles in type-S cells. The findings that type-A and type-S synoviocytes show distinctly different organization of the lysosomal system indicate that the roles of the lysosomes in these two types of cells may be different.     

The role of intraorganellar Ca(2+) in late endosome-lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles

We have investigated the requirement for Ca(2+) in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca(2+). Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca(2+) reduced the amount of fusion. The requirement for Ca(2+) for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome-lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca(2+) plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway.     

A novel method of imaging lysosomes in living human mammary epithelial cells

Cancer cells invade by secreting degradative enzymes which, under normal conditions, are sequestered in lysosomal vesicles. The ability to noninvasively label lysosomes and track lysosomal trafficking would be extremely useful to understand the mechanisms by which degradative enzymes are secreted in the presence of pathophysiological environments, such as hypoxia and acidic extracellular pH, which are frequently encountered in solid tumors. In this study, a novel method of introducing a fluorescent label into lysosomes of human mammary epithelial cells (HMECs) was evaluated. Highly glycosylated lysosomal membrane proteins were labeled with a newly synthesized compound, 5-dimethylamino-naphthalene-1-sulfonic acid 5-amino-3,4,6-trihydroxy-tetrahydro-pyran-2-ylmethyl ester (6-O-dansyl-GlcNH2). The ability to optically image lysosomes using this new probe was validated by determining the colocalization of the fluorescence from the dansyl group with immunofluorescent staining of two well-established lysosomal marker proteins, LAMP-1 and LAMP-2. The location of the dansyl group in lysosomes was also verified by using an anti-dansyl antibody in Western blots of lysosomes isolated using isopycnic density gradient centrifugation. This novel method of labeling lysosomes biosynthetically was used to image lysosomes in living HMECs perfused in a microscopy-compatible cell perfusion system.     

Tubular lysosomes in ruffle-ended ameloblasts associated with enamel maturation in rat incisor

Trimetaphosphatase (TMPase) and cytidine-5'-monophosphatase (CMPase) were localized to investigate the lysosomal system, particularly tubular lysosomes, in ruffle-ended ameloblasts associated with maturation of enamel in rat incisor. Demineralized specimens were incubated for TMPase and for CMPase in a modified medium where cerium was used as the capture ion. Ruffle-ended ameloblasts showed distal invaginations and membrane-bound bodies filled with fine granular material, some of which displayed CMPase reaction product. Elongated tubular configurations 80-140 nm wide were distributed throughout the cytoplasm and were reactive with both TMPase and CMPase, thus characterizing these structures as lysosomes. They often contained fine granular material morphologically similar to that present in multivesicular bodies. During late enamel maturation, fewer tubular lysosomes were observed when compared to early maturation. These cytochemical results demonstrate the presence of tubular lysosomes in ruffle-ended ameloblasts, and it is suggested that they are elements of the endosomal system in these cells. These findings are also consistent with a resorptive function for ruffle-ended ameloblasts during enamel maturation.     

Secretory lysosome biogenesis in cytotoxic T lymphocytes from normal and Chediak Higashi syndrome patients

The lytic proteins mediating target cell killing are stored in the lysosomes of activated cytotoxic T lymphocytes (CTL) and are secreted upon recognition of a target cell. These secretory lysosomes cannot be detected in resting T lymphocytes. Interaction of a resting cell with a target cell activates de novo formation of secretory lysosomes. CTL clones in culture mimic this behaviour, and so provide an ideal system for studying secretory lysosome biogenesis and maturation. In the genetic disease, Chediak Higashi syndrome (CHS), all lysosomes in the cells are enlarged and reduced in number compared with wild-type (WT) cells. We have used CTL from this disease to study secretory lysosome biogenesis and maturation. We show that at early stages after activation the secretory lysosomes are identical in WT and mutant cells, and that delivery of proteins to the secretory lysosome along the biosynthetic and endocytic pathways is normal in the mutant cells. With time, the lysosomes in the mutant cells aggregate, become larger and fewer in number and eventually form giant structures. Our results show that the initial steps of secretory lysosome formation are normal in CHS, but that the organelles subsequently fuse together during cell maturation to form the giant secretory lysosomes.     

Characterization of a phosphate transport system in human fibroblast lysosomes.

The uptake of [32P]KH2PO4 by Percoll-purified human fibroblast lysosomes at pH 7.0 was investigated to determine if lysosomes contain a transport system recognizing phosphate. Lysosomal phosphate uptake was linear for the first 2 min, attained a steady state by 8-10 min at 37 degrees C, and was not Na+ or K+ dependent. Upon entering lysosomes, [32P]phosphate was rapidly metabolized to trichloroacetic acid-soluble and trichloroacetic acid-insoluble products. After 1-min incubations, 50% of the radioactivity recovered from lysosomes was in the form of inorganic phosphate; and after a 2.5-min incubation, 27% of the radioactivity was recovered as inorganic phosphate. When lysosomes are loaded with radioactivity by incubation with 0.03 mM [32P]KH2PO4 for 25 min and then washed at 4 degrees C, lysosomes fail to release the accumulated radioactivity during a subsequent incubation at 37 degrees C. Lysosomal phosphate uptake gave linear Arrhenius plots (Q10 = 1.8) and was inversely proportional to medium osmolarity. Phosphate uptake was maximal at pH 5-6, half-maximal at pH 7.1, with little transport activity at pH greater than 8, suggesting that the transport system recognizes the monobasic form of phosphate. Lysosomal phosphate uptake is saturable, displaying a Km of 5 microM at pH 7.0 and 37 degrees C. High specificity for phosphate is demonstrated since large concentrations of Na2SO4, NaHCO3, KCl, NaCl, 5'-AMP, or the anion transport inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, have no effect on lysosomal phosphate transport. In contrast, the phosphate analog, arsenate, strongly inhibits lysosomal phosphate uptake in a competitive manner with a Ki of 7 microM. Pyridoxal phosphate, CTP, adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP), and glucose 6-phosphate were found to be noncompetitive inhibitors of lysosomal phosphate uptake displaying Ki values of 80-250 microM. When lysosomes are incubated with [gamma-32P]ATP, the lysosomal membrane ATPase hydrolyzes the ATP to form inorganic phosphate which then enters lysosomes by this lysosomal phosphate transport route.     

Isolation of rat liver lysosomes by a single two-phase partition on dextran/polyethylene glycol.

Crude mitochondria from liver rats were added to a two-phase system containing dextran and polyethylene glycol. The polymer and ionic concentration values of the two-phase system were changed in order to separate lysosomes from mitochondria. The best separation of lysosomes and mitochondria was obtained at 6.6-6.6% (w/w) dextran-polyethylene glycol and 5 mmol/kg ammonium chloride as shown by enzyme assays. This procedure showed good reproducibility, and lysosomes were never contaminated with more than 16% mitochondria, as determined by succinate dehydrogenase activity, and beta-D-galactosidase and acid phosphatase activities were enriched five- to sixfold. The lipid composition profile of lysosomes was quite similar to that obtained by means of free carrier electrophoresis, considered a reference method.     

Morphometric analysis of the autophagic and crinophagic lysosomal systems in mammotropes throughout the estrous cycle of the rat

Summary The crinophagic and autophagic lysosomal systems were studied in mammotropes (prolactin secreting cells) of the adenohypophysis throughout the estrous cycle of the rat. By means of morphometric analysis, it was found that the volume of secondary autophagic lysosomes was usually greater than that of the crinophagic type. Although the volumes of both secondary autophagic and crinophagic lysosomes were minimal throughout proestrus and diestrus 2, the autophagic lysosomal volume per mammotrope was elevated during the estrous period. The volume of secondary crinophagic lysosomes per mammotrope increased during late estrus and remained elevated throughout early diestrus 1. Furthermore, there was an inverse relationship between the volume of mature secretory granules per cell and of the crinophagic system. These data suggest a role for lysosomes in the regulation of synthesis and secretion of prolactin by the adenohypophysis of the rat.Supported by grant HD 11571 from the National Institutes of Health     

Thapsigargin distinguishes membrane fusion in the late stages of endocytosis and autophagy

A close relationship exists between autophagy and endocytosis with both sharing lysosomes as their common end-point. Autophagy even requires a functional endocytic pathway. The point at which the two pathways merge, i.e., fusion of autophagosomes and endosomes with lysosomes is poorly understood. Early work in yeast and more recent studies in mammalian cells suggested that conventional membrane trafficking pathways control the fusion of autophagosomes with lysosomes; Rab GTPases are required to recruit tethering proteins which in turn coordinate the SNARE family of proteins that directly drive membrane fusion. Some components required for endosomes to fuse with lysosomes are also shared by autophagosomes; both are thought to require the GTPase Rab7 and the homotypic fusion and vacuole protein sorting (HOPS) complex. Essentially, the autophagosome becomes endosome-like, allowing it to recruit the common fusion machinery to deliver its contents to the lysosome. This raises an interesting question of how the cell determines when the autophagosome is ready to fuse with the endocytic system and bestows upon it the properties required to recruit the fusion machinery. Our recent work has highlighted this conundrum and shown that autophagosome fusion with lysosomes has specific distinctions from the parallel endosomal-lysosomal pathway.     

Thapsigargin distinguishes membrane fusion in the late stages of endocytosis and autophagy

A close relationship exists between autophagy and endocytosis with both sharing lysosomes as their common end-point. Autophagy even requires a functional endocytic pathway. The point at which the two pathways merge, i.e., fusion of autophagosomes and endosomes with lysosomes is poorly understood. Early work in yeast and more recent studies in mammalian cells suggested that conventional membrane trafficking pathways control the fusion of autophagosomes with lysosomes; Rab GTPases are required to recruit tethering proteins which in turn coordinate the SNARE family of proteins that directly drive membrane fusion. Some components required for endosomes to fuse with lysosomes are also shared by autophagosomes; both are thought to require the GTPase Rab7 and the homotypic fusion and vacuole protein sorting (HOPS) complex. Essentially, the autophagosome becomes endosome-like, allowing it to recruit the common fusion machinery to deliver its contents to the lysosome. This raises an interesting question of how the cell determines when the autophagosome is ready to fuse with the endocytic system and bestows upon it the properties required to recruit the fusion machinery. Our recent work has highlighted this conundrum and shown that autophagosome fusion with lysosomes has specific distinctions from the parallel endosomal-lysosomal pathway.     

Reconstitution of an endosome-lysosome interaction in a cell-free system

A cell-free model for the transfer of endocytosed material to lysosomes is described. Rat liver late endosomes, loaded in vivo with radiolabeled ligand by intravenous injection shortly before killing the animal, showed a specific interaction with lysosomes when incubated at 37 degrees C in the presence of cytosol and an ATP regenerating system. The location of the ligand, generally asialofetuin, was analyzed by isopycnic centrifugation on Nycodenz gradients. Appearance of radiolabel in the lysosomal position on such gradients was maximal after approximately 30 min at 37 degrees C and required the provision of undamaged cytosol, lysosomes, and an ATP regenerating system. It could not be accounted for by nonspecific bulk aggregation of membranes. Transfer occurred only from late endosomes; radiolabel in early endosomes was unaffected. Digestion of the asialofetuin, as shown by the appearance of TCA-soluble radioactivity, occurred on incubation at 37 degrees C and was increased by the provision of an ATP regenerating system.     

Ultrastructural localization of polysaccharides in the vacuolar system of an established cell line

Polysaccharides in the vacuolar system of an established line of monkey kidney epithelial cells, were investigated by high resolution radioautography following incorporation of 3H-fucose and by the periodic acid-thiosemicarbazide-silver proteinate staining method. Significant amounts of glycogen and glycoproteins were found in different lysosomes and prelysosomes. The radioautographic labeling of these substances correlated with the cytochemical observations. Analysis of the 3H-fucose pattern of labeling suggests that glycoproteins are transported through the different components of the vacuolar system of the cell in a sequential fashion rather than via independent pathways. The possible functional significance of polysaccharides in lysosomes is discussed. It is suggested that glycogen is taken up by lysosomes through autophagic segregation and through infolding and vesiculation of the lysosomal surface, and that the majority of glycoprotein in the lysosomal membranes does not have acid hydrolase activity.     

The endosome-lysosome system in the absorptive cells of goldfish hindgut

Summary The vacuolar system in the absorptive cells of the goldfish hindgut was studied by rapid freeze-substituted and cytochemical techniques. The apical cytoplasm of the absorptive cells contained two types of vacuoles: endosomes and lysosomes. The former were characterized by an absence of acid phosphatase activity, a dot-like distribution of material at the peripheral rim, the labelling of the inner surface with horseradish peroxidase (HRP), and by frequent connections to cytoplasmic tubules (CT), which were also free of acid phosphatase activity. The latter vacuole was preferentially located in the deeper cytoplasm and was characterized by the presence of acid phosphatase activity, an electron-dense interior matrix, a peripheral electron-lucent region (a halo), and by the detachment of HRP from the inner surface. Connections between CTs and these latter vacuoles were rarely seen. In the deeper cytoplasm, fusion between endosomes and lysosomes was sometimes observed. These results suggest that the vacuoles which are associated with CTs are endosomes, but not lysosomes, and that internalized materials are transported through the endosome-lysosome system to a giant food vacuole in the cell.     

A new method for the preparation of rat liver lysosomes. Separation of cell organelles of rat liver by carrier-free continuous electrophoresis

A combination of differential centrifugation and carrier-free continuous electrophoresis is introduced as a new method for the isolation of animal cell organelles. Various buffers were systematically checked in order to find the system which preserves the organelles and gives as well a good separation in the free-flow electrophoresis apparatus. Triethanolamine-acetate buffer (10 mM), pH 7.4 was used. The isolated lysosomes were pure according to marker enzymes and electron micrographs. A heterogeneity of the lysosomes in electrophoretic mobility was demonstrated with respect to the marker enzymes arylsulfatase and β-glucuronidase. The lysosomes with higher mobility showed a maximum enrichment of 240-fold with respect to arylsulfatase. The lysosomes with lower electrophoretic mobility showed a 65-fold enrichment with respect to β-glucuronidase. The ratio of β-glucuronidase to arylsulfatase varied from 2:1 to 1:2 in lysosomes of different mobility. The yield amounted to approximately 1 mg of lysosomal protein per gram of liver protein. 5–8 mg of lysosomes can be obtained in one experiment. The electrophoretic separation proves to be an effective tool in obtaining pure and well preserved lysosomes.     

Degradation of aspartate aminotransferase in rat liver lysosomes

Highly purified lysosomes from the normal and leupeptin-treated rat livers were subjected to immunoblot analysis using antibodies against cytosolic and mitochondrial isozymes of aspartate aminotransferase (cAspAT and mAspAT). In the case of cAspAT (subunit M.W. = 46K), the leupeptin-treated lysosomes showed a major band of 46K and a minor band of 36K while normal lysosomes showed a major band of 36K and a minor band of 41K. In the case of mAspAT (subunit M.W. = 44K), the leupeptin-treated lysosomes showed a 44K band and the normal lysosomes showed a 40K band. These observations suggest that both cAspAT and mAspAT are sequestered into lysosomes with the original subunit molecular weights and are degraded in the lysosomes by way of sequential formation of relatively stable intermediates with distinct molecular weights.     

The morphology but not the function of endosomes and lysosomes is altered by brefeldin A

Brefeldin A (BFA) induces the formation of an extensively fused network of membranes derived from the trans-Golgi network (TGN) and early endosomes (EE). We describe in detail here the unaffected passage of endocytosed material through the fused TGN/EE compartments to lysosomes in BFA-treated cells. We also confirmed that BFA caused the formation of tubular lysosomes, although the kinetics and extent of tubulation varied greatly between different cell types. The BFA-induced tubular lysosomes were often seen to form simple networks. Formation of tubular lysosomes was microtubule-mediated and energy-dependent; interestingly, however, maintenance of the tubulated lysosomes only required microtubules and was insensitive to energy poisons. Upon removal of BFA, the tubular lysosomes rapidly recovered in an energy-dependent process. In most cell types examined, the extensive TGN/EE network is ephemeral, eventually collapsing into a compact cluster of tubulo-vesicular membranes in a process that precedes the formation of tubular lysosomes. However, in primary bovine testicular cells, the BFA-induced TGN/EE network was remarkably stable (for > 12 h). During this time, the TGN/EE network coexisted with tubular lysosomes, however, the two compartments remained completely separate. These results show that BFA has multiple, profound effects on the morphology of various compartments of the endosome-lysosome system. In spite of these changes, endocytic traffic can continue through the altered compartments suggesting that transport occurs through noncoated vesicles or through vesicles that are insensitive to BFA.