The values and limits of an in vitro model of Pompe disease: The best laid schemes o’ mice an’ men ……..†

In Pompe disease, a lysosomal glycogen storage disorder, cardiac and skeletal muscle abnormalities are responsible for premature death and severe weakness. Swollen glycogen-filled lysosomes, the expected pathology, are accompanied in skeletal muscle by a secondary pathology – massive accumulation of autophagic debris – that appears to contribute greatly to the weakness. We have tried to reproduce these defects in murine, Pompe myotubes derived from either primary myoblasts or myoblasts with extended proliferative capacity. The cells accumulated large lysosomes filled with glycogen, but, to our disappointment, did not have autophagic buildup even though basal autophagy was intact. When we suppressed autophagy by knocking down Atg7, we found that glycogen uptake by lysosomes was not affected, suggesting that macroautophagy is not the major pathway for glycogen delivery to lysosomes. But two apparently incidental observations – a peculiar distribution of both microinjected dextran and of small acidic structures adjacent to the interior membrane of large alkalinized glycogen-containing lysosomes – raised the possibility that glycogen traffics to the lysosomes by microautophagy or/and by the engulfment of small lysosomes by large ones. The cultured myotubes, therefore, appear to be a useful model for studying the mechanisms involved in glycogen accumulation in Pompe disease and to test substrate deprivation approaches.

^?From To a Mouse, Robert Burns, 1785     

Changes in glucose and lipid metabolism in starved or starved-refed Japanese quail (coturnix coturnix japonica) in relation to fine structure of liver cells

1. Metabolic response of adult quail to fasting or refeeding was studied by measuring the main blood and hepatic metabolites. Moreover, the fine structure of hepatocytes in these physiological conditions was described. 2. Starvation or refeeding did not affect glycemia in male as in female quails. 3. Fasting had no effect on plasma free fatty acids in female quails, whereas plasma triglycerides were markedly decreased. 4. In fasted quails, there was an active ketogenesis with a high 3-hydroxybutyrate/acetoacetate ratio. 5. Ultrastructural aspect of liver parenchymal cells from fasted quails revealed alterations in the quantity of glycogen, smooth endoplasmic reticulum, lysosomes and in the form of the rough endoplasmic reticulum. 6. The significance of these morphological changes was discussed in relation to an hormonal stimulation.     

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

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

An unusual ultrastructural association of smooth membranes and glycogen particles: The glycogen body

Summary An electron microscope study of the epithelium of rabbit fallopian tube demonstrated a rarely described intracytoplasmic structure consisting of an array of smooth membranes associated with glycogen particles. This organelle is seen exclusively in the ciliated cells. A three-dimensional reconstruction of these glycogen bodies has been made from serial sections. The peripheral localization of the rough-surfaced membranes in continuity with intra-corpuscular smooth membranes, which have lost their granules, suggests a possible role for the rough membranes in the genesis of the smooth membranes of these glycogen bodies. The role of both the smooth and the rough membranes in glycogenesis and glycogenolysis is discussed.This investigation was made in part in the Laboratoire d'Hormonologie et de Cytologie Expérimentale, Hôpital Broca, Paris.     

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.     

Autophagy–mediated plasma membrane removal promotes the formation of epithelial syncytia

Epithelial wound healing in Drosophila involves the formation of multinucleate cells surrounding the wound. We show that autophagy, a cellular degradation process often deployed in stress responses, is required for the formation of a multinucleated syncytium during wound healing, and that autophagosomes that appear near the wound edge acquire plasma membrane markers. In addition, uncontrolled autophagy in the unwounded epidermis leads to the degradation of endo‐membranes and the lateral plasma membrane, while apical and basal membranes and epithelial barrier function remain intact. Proper functioning of TORC1 is needed to prevent destruction of the larval epidermis by autophagy, in a process that depends on phagophore initiation and expansion but does not require autophagosomes fusion with lysosomes. Autophagy induction can also affect other sub‐cellular membranes, as shown by its suppression of experimentally induced laminopathy‐like nuclear defects. Our findings reveal a function for TORC1‐mediated regulation of autophagy in maintaining membrane integrity and homeostasis in the epidermis and during wound healing.     

Extracellular shedding of photoreceptor membrane in the open rhabdom of a tipulid fly

Summary The compound eyes of the Australian tipulid fly, Ptilogyna, shed the bulk of their rhabdomeral membrane to extracellular space during turnover. The rhabdomeres of the retinulae lie in a common extracellular space (ECS), which is subdivided in the proximal retina. Before dawn, a distal region of the microvilli in each rhabdomere differentiates and becomes less electron-dense after conventional fixation. The differentiated region then dilates and develops an irregular profile. A few hours after dawn, the transformed tips break off and form a detritus in the ECS. The degraded membrane is internalised back into the retinula cells by mass endocytosis. Retinulae develop pseudopodia at sites bordering the ECS and engulf the membrane detritus, which comes to lie first of all in vacuoles within the receptor cells and then forms very large multivesicular bodies. The latter transform to multilamellar and residual bodies and are, presumably, lysed. Surrounding these secondary lysosomes are rough endoplasmic reticulum and smooth tubular systems, tentatively considered on comparative grounds to provide hydrolases. The literature concerning the ultrastructure of compound eyes offers a small number of instances where extracellular shedding can be suspected for morphological reasons. Attention is drawn to analogies with the shedding of photoreceptor membranes in vertebrate retinae.     

Highly purified plasma membranes from rat hepatocytes following rate-isopycnic zonal centrifugation

Plasma membranes from liver parenchymal cells were isolated by rate-isopycnic zonal centrifugation. A method is described for the Beckman size 15 zonal rotor. It involved preparation from a perfused liver of a parenchymal cell-enriched homogenate in isoosmotic sucrose. The nuclear fraction containing membranes was recovered by centrifugation. The resuspended pellet was applied on the gradient of the zonal rotor. The isolated membranes had the same isopycnic banding density as 37% sucrose (w/w). The specific activity of 5′-nucleotidase, a widely used plasma membrane marker, was 105 μmoles·(mg protein)^?1·h^?1 being enriched by a factor of 50 as compared with parenchymal cell homogenate. The plasma membrane fraction was free of the mitochondrial and lysosomal enzymes, succinate dehydrogenase and acid phosphatase. No DNA and 10 μg RNA per mg plasma membrane protein were found. The purity of the membranes and their morphological appearance were controlled by electron microscopy. The preparation consisting of large membrane sheets showed a considerable purification away from other cellular components. A comparison with similar methods indicates that plasma membranes of a higher degree of purity can be obtained from parenchymal cells.     

Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder—murine Pompe disease

Autophagy, an intracellular system for delivering portions of cytoplasm and damaged organelles to lysosomes for degradation/recycling, plays a role in many physiological processes and is disturbed in many diseases. We recently provided evidence for the role of autophagy in Pompe disease, a lysosomal storage disorder in which acid alpha-glucosidase, the enzyme involved in the breakdown of glycogen, is deficient or absent. Clinically the disease manifests as a cardiac and skeletal muscle myopathy. The current enzyme replacement therapy (ERT) clears lysosomal glycogen effectively from the heart but less so from skeletal muscle. In our Pompe model, the poor muscle response to therapy is associated with the presence of pools of autophagic debris. To clear the fibers of the autophagic debris, we have generated a Pompe model in which an autophagy gene, Atg7, is inactivated in muscle. Suppression of autophagy alone reduced the glycogen level by 50–60%. Following ERT, muscle glycogen was reduced to normal levels, an outcome not observed in Pompe mice with genetically intact autophagy. The suppression of autophagy, which has proven successful in the Pompe model, is a novel therapeutic approach that may be useful in other diseases with disturbed autophagy.Key words: Pompe disease, lysosomal glycogen storage, myopathy, Atg7, enzyme replacement therapy     

Long-term live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms

Plasmodium parasites are transmitted by Anopheles mosquitoes to the mammalian host and actively infect hepatocytes after passive transport in the bloodstream to the liver. In their target host hepatocyte, parasites reside within a parasitophorous vacuole (PV). In the present study it was shown that the parasitophorous vacuole membrane (PVM) can be targeted by autophagy marker proteins LC3, ubiquitin, and SQSTM1/p62 as well as by lysosomes in a process resembling selective autophagy. The dynamics of autophagy marker proteins in individual Plasmodium berghei-infected hepatocytes were followed by live imaging throughout the entire development of the parasite in the liver. Although the host cell very efficiently recognized the invading parasite in its vacuole, the majority of parasites survived this initial attack. Successful parasite development correlated with the gradual loss of all analyzed autophagy marker proteins and associated lysosomes from the PVM. However, other autophagic events like nonselective canonical autophagy in the host cell continued. This was indicated as LC3, although not labeling the PVM anymore, still localized to autophagosomes in the infected host cell. It appears that growing parasites even benefit from this form of nonselective host cell autophagy as an additional source of nutrients, as in host cells deficient for autophagy, parasite growth was retarded and could partly be rescued by the supply of additional amino acid in the medium. Importantly, mouse infections with P. berghei sporozoites confirmed LC3 dynamics, the positive effect of autophagy activation on parasite growth, and negative effects upon autophagy inhibition.     

Hydrogenosome autophagy: an ultrastructural and cytochemical study.

The process of autophagy was studied in Tritrichomonas foetus under serum deprivation, drug treatment (hydroxyurea, zinc sulfate), and also in normal conditions using routine electron microscopy, freeze-fracture, freeze-substitution, and enzyme cytochemistry. We also used gold particles conjugated with bovine albumin to better characterize the participation of lysosomes in the process of hydrogenosome degradation. Apparently normal hydrogenosomes and also giant, abnormal hydrogenosomes presenting internal membranes were seen in the autophagic process. The first event observed was the rough endoplasmic reticulum surrounding and enclosing the hydrogenosome, forming an isolation membrane. The hydrogenosomes were first sequestered from the remaining cytoplasm and then degraded within lysosomes. The autophagic vacuoles were limited by double or multiple concentric membranes and many contained recognizable hydrogenosomes, probably in the preliminary steps of degradation. Lysosomes seemed to fuse with autophagic vacuoles forming a degradative structure bound by a single membrane and containing hydrogenosomes in various stages of degeneration. Hydrogenosomes appeared partially degraded, forming hydrogenosomal remnants. It was observed that there is a removal of hydrogenosomes in normal cells and in cases of cell toxicity.     

Schistosoma mansoni: Ultrastructure of early transformation of skin- and shear-pressure-derived schistosomules

Against the background of cercarial fine structure, ultrastructural changes were compared in schistosomules of Schistosoma mansoni 30 min and 1 hr after their production in vivo by skin penetration and in vitro by shear pressure. The same developmental pattern was observed in schistosomules of both derivations. In vitro schistosomules, however, developed more slowly, resembled cercariae more closely, and varied less among organisms than did in vivo schistosomules. The greatest morphological changes were observed in the 1-hr in vivo schistosomules. These were as follows: (1) in tegument, formation of transient microvilli, a hepatalaminate outer membrane and accented surface invaginations, loss of glycocalyx, movement outward of cyton vesicles via bridges, accumulation of multilaminate bodies around bridge openings; (2) in the anterior organ (oral sucker), movement of head gland vesicles via the ducts into tegument followed by collapse of the gland fundus, disappearance of the circumfundal cells and two large support cells, and the appearance in these areas of membranes and parenchymal cells; (3) secretion of the acetabular gland contents, collapse of the glands and replacement by membranes and parenchymal cells; (4) peristaltic activity of the digestive tract as shown by alternate areas of lumen constriction and dilation; (5) loss of bladder and contraction of the small aboral collecting tubules; and (6) conversion of heterochromatic parenchymal cell nuclei to euchromatic. In contrast, the 1-hr in vitro shear schistosomules resembled 30-min in vivo schistosomules, retaining many cercarial features.     

Autophagy and acid phosphatase activity in the corpora allata of adult mated females of Diploptera punctata

The corpora allata exbibit cycles of synchronous cell growth and atrophy during ovarian cycles in adult females of the cockroach Diploptera punctata. In the present report, the process of synchronous autophagy of organelles which results in cellular atrophy was investigated. In general, unwanted organelles were sequentially sequestered by several different mechanisms and then targeted for destruction. Autophagy was initiated on day 4 when corpus allatum cells were largest and most actively synthesizing juvenile hormone. The first sign of the initiation of autophagy was aggregation of ribosomes in an isolation membrane. By day 5, many organelles were isolated in the autophagic vacuoles. The ribosomecontaining vacuoles were wrapped by flattened stacks of Golgi cisternae to form conspicuous whorl-like autophagosomes. This is a previously undescribed type of autophagic vacuole with the entire complex of Golgi cisternae forming part of the autophagic membranes. Smooth endoplasmic reticulum was wrapped into membranous autophagic vacuoles with concentric arrays of doubel membranes. Plasma membrane was invaginated and then isolated in a multivesicular body. These three different types of isolated vacuoles did not show acid phosphatase activity as indicated by histochemical staining with -glycerophosphate as substrate. Subsequently, these autophagosomes fused with each other and with 1° or 2° lysosomes to form giant autophagolysosomes. Some mitochondria appeared to have coalesced directly into autophagolysosomes. Golgi complexes were evident during this period; they actively participated in making lysosomal enzymes. Cytoskeletons were frequently observed in the vicinity of autophagic vacuoles and were presumably involved in the transport of the vacuoles. As a result of lysosomal degradation lipofuscins and dense bodies were frequently observed by days 9–12 indicating atrophy of corpus allatum cells. Structural parameters, especially those present early in autophagy, such as the isolation membrane, ribosome-containing vacuoles and whorl-like autophagosomes, can be used to search for potential growth regulators responsible for the induction of autophagy, of the corpora allata, and the subsequent termination in juvenile hormone synthesis.     

Starch Binding Domain-containing Protein 1/Genethonin 1 Is a Novel Participant in Glycogen Metabolism

Stbd1 is a protein of previously unknown function that is most prevalent in liver and muscle, the major sites for storage of the energy reserve glycogen. The protein is predicted to contain a hydrophobic N terminus and a C-terminal CBM20 glycan binding domain. Here, we show that Stbd1 binds to glycogen in vitro and that endogenous Stbd1 locates to perinuclear compartments in cultured mouse FL83B or Rat1 cells. When overexpressed in COSM9 cells, Stbd1 concentrated at enlarged perinuclear structures, co-localized with glycogen, the late endosomal/lysosomal marker LAMP1 and the autophagy protein GABARAPL1. Mutant Stbd1 lacking the N-terminal hydrophobic segment had a diffuse distribution throughout the cell. Point mutations in the CBM20 domain did not change the perinuclear localization of Stbd1, but glycogen was no longer concentrated in this compartment. Stable overexpression of glycogen synthase in Rat1WT4 cells resulted in accumulation of glycogen as massive perinuclear deposits, where a large fraction of the detectable Stbd1 co-localized. Starvation of Rat1WT4 cells for glucose resulted in dissipation of the massive glycogen stores into numerous and much smaller glycogen deposits that retained Stbd1. In vitro, in cells, and in animal models, Stbd1 consistently tracked with glycogen. We conclude that Stbd1 is involved in glycogen metabolism by binding to glycogen and anchoring it to membranes, thereby affecting its cellular localization and its intracellular trafficking to lysosomes.     

Autophagy of metabolically inert substances injected into fibroblasts in culture

We have examined the morphologic characteristics of fibroblasts cultured from the beige mouse, a genetic variant phenotypically similar to human Chediak-Higashi syndrome (CHS). These cultured fibroblasts are characterized by large, amorphous dense body inclusions in their cytoplasm, often as large as the cell nucleus. Using time-lapse video phase-contrast microscopy, we have observed the formation of these large dense bodies through fusion of relatively normal-appearing lysosomes in the beige mouse fibroblast. After formation of these large inclusions, cells occasionally extruded the contents of these structures through apparent fusion with the plasma membrane and rapid exocytosis. Fibroblasts cultured from normal black mice showed no evidence of fusion between lysosomes or exocytosis of lysosomes. However, the uptake of extracellular medium through macropinocytosis, subsequent actions of lysosomes on these macropinosomes through saltatory motion, cellular migration and ruffling activity appeared normal in beige mouse fibroblasts. Immunocytochemical localization of α₂-macroglobulin, a normal serum protein commonly incorporated into lysosomes in cultured fibroblasts by receptor-mediated endocytosis, showed that the large dense bodies contained α₂-macroglobulin, in keeping with their lysosomal origin. This suggested further that receptor-mediated endocytosis in these cells was relatively normal. In addition, light and electron microscopic cytochemistry showed these large inclusions to be acid-phosphatase positive, further characterizing them as lysosomal. The electron microscopic appearance of these dense inclusions was consistent with their origin through repeated fusion of lysosomes. It is suggested that a primary defect in this disease may be the ability of mature lysosomal membranes to fuse, unlike normal lysosomal membranes, indicating perhaps an alteration in some specific component of the lysosomal membranes in CHS.     

Distribution of the integral membrane protein NADH-cytochrome b5 reductase in rat liver cells, studied with a quantitative radioimmunoblotting assay.

The intracellular localization of the post-translationally inserted integral membrane protein, NADH-cytochrome b5 reductase, was investigated, using a quantitative radioimmunoblotting method to determine its concentration in rat liver subcellular fractions. Subcellular fractions enriched in rough or smooth microsomes, Golgi, lysosomes, plasma membrane and mitochondrial inner or outer membranes were characterized by marker enzyme analysis and electron microscopy. Reductase levels were determined both with the NADH-cytochrome c reductase activity assay, and by radioimmunoblotting, and the results of the two methods were compared. When measured as antigen, the reductase was relatively less concentrated in microsomal subfractions, and more concentrated in fractions containing outer mitochondrial membranes, lysosomes and plasma membrane than when measured as enzyme activity. Rough and smooth microsomes had 4-5-fold lower concentrations, on a phospholipid basis than did mitochondrial outer membranes. Fractions containing Golgi, lysosomes and plasma membrane had approximately 14-, approximately 16, and approximately 9-fold lower concentrations of antigen than did mitochondrial outer membranes, respectively, and much of the antigen in these fractions could be accounted for by cross-contamination. No enzyme activity or antigen was detected in mitochondrial inner membranes. Our results indicate that the enzyme activity data do not precisely reflect the true enzyme localization, and show an extremely uneven distribution of reductase among different cellular membranes.     

ELECTRON MICROSCOPY OF THE OXYNTIC CELL IN THE GASTRIC GLANDS OF THE BULLFROG (RANA CATESBIANA) : I. The Non-Acid-Secreting Gastric Mucosa

The fine structure of the oxyntic cell from the gastric glands of the bullfrog was studied in lead hydroxide—stained sections of gastric mucosa fixed in buffered osmium tetroxide and embedded in n-butyl methacrylate. The oxyntic cell in non-acid-secreting stomachs (gastric juice pH, 7.4–7.8) is characterized by: (a) numerous closely packed smooth surfaced vesicular and tubular profiles disposed randomly in the cell; some of these elements show interconnections making it possible to identify this component with smooth surfaced endoplasmic reticula of certain other cell types, (b) a small percentage of rough surfaced profiles characteristic of endoplasmic reticula possessing RNP particles on the outer membrane surfaces, (c) a Golgi complex consisting of multiple isolated non-polarized arrays of smooth surfaced parallel elongated profiles and associated vesicular elements, (d) a sparse granular component (140 A) scattered freely in the cytoplasmic matrix, (e) numerous mitochondria with a dense matrix and containing an unusually large number of closely approximated cristae, (f) a number of zymogen granules consisting of either a dense body limited by a membrane or surrounded by a halo of less dense material which is in turn limited by a membrane, and (g) a number of granules (~260 A) containing several smaller granules (~80 A) identified presumably as glycogen. Intracellular canaliculi were not observed. Instead the free surface of the oxyntic cell facing the lumen of the gastric gland shows a complicated plication of the plasma membrane. Intercellular canaliculi are seen frequently between adjacent oxyntic cells. The walls of these canaliculi are made up of folded and ruffled cell membranes. The basal surface of the cell also exhibited this type of configuration. Occasional smooth surfaced profiles are seen communicating with the free surface, the wall of an intercellular canaliculus, or the basal surface of the cell. Although nerve endings were not found in association with oxyntic cells, unmyelinated nerves were observed in the vicinity of the gastric glands.     

Studies on vinblastine-induced autophagocytosis in mouse liver. II. Origin of membranes and acquisition of acid phosphatase

The origin of the membranes of autophagic vacuoles (AV) and acquisition of acid phosphatase into AV's were studied in vinblastine-induced autophagocytosis (VBL, 50 mg/kg, i.p.) in mouse hepatocytes. Using unbuffered OsO4, very intense staining was observed in the outer cisternae of the Golgi apparatus and also frequently in the cavity between the double membranes obviously destined to form AV's as well as in the cavity between the double membranes of newly formed AV's. There may occur a transformation process in the membranes limiting an AV analogous to that observed at the Golgi cisternae. The transformation of the outer AV membrane occurs independently of fusion with lysosomes. Inosine diphosphatase activity was localized within the cisternae and on the membranes of the endoplasmic recticulum and occasionally within the innermost cisterna of the Golgi apparatus. The results together with the unbuffered OsO4-staining pattern suggest that the membranes of most AV's are derived from the transformed smooth surfaced cisternae of the endoplasmic reticulum which do not have inosine diphosphatase activity. Acid phosphatase activity was localized in lysosomes, occasionally within the innermost cisternae of the Golgi apparatus, between the double membranes of a few newly formed AV's and within most older single membranes of a few newly formed AV's and within most older single membrane-limited AV's. VBL did not prevent the fusion of lysosomes with AV's.     

The role of ESCRT proteins in fusion events involving lysosomes, endosomes and autophagosomes

ESCRT (endosomal sorting complex required for transport) proteins were originally identified for their role in delivering endocytosed proteins to the intraluminal vesicles of late-endosomal structures termed multivesicular bodies. Multivesicular bodies then fuse with lysosomes, leading to degradation of the internalized proteins. Four ESCRT complexes interact to concentrate cargo on the endosomal membrane, induce membrane curvature to form an intraluminal bud and finally pinch off the bud through a membrane-scission event to produce the intraluminal vesicle. Recent work suggests that ESCRT proteins are also required downstream of these events to enable fusion of multivesicular bodies with lysosomes. Autophagy is a related pathway required for the degradation of organelles, long-lived proteins and protein aggregates which also converges on lysosomes. The proteins or organelle to be degraded are encapsulated by an autophagosome that fuses either directly with a lysosome or with an endosome to form an amphisome, which then fuses with a lysosome. A common machinery is beginning to emerge that regulates fusion events in the multivesicular body and autophagy pathways, and we focus in the present paper on the role of ESCRT proteins. These fusion events have been implicated in diseases including frontotemporal dementia, Alzheimer's disease, lysosomal storage disorders, myopathies and bacterial pathogen invasion, and therefore further examination of the mechanisms involved may lead to new insight into disease pathogenesis and treatments.     

Formation of triton WR 1339-filled rat liver lysosomes : II. Involvement of autophagy and of pre-existing lysosomes

The participation of endocytosis in the formation of Triton-filled lysosomes was followed after injecting Triton WR 1339 simultaneously with colloidal gold or horseradish peroxidase. According to cytomorphometric measurements Triton WR 1339 also significantly enhances autophagy. The analysis of the influence of Triton WR 1339 subfractions shows that autophagy is only augmented by the polymer component (‘macrotriton’) but not by the low molecular component (‘microtriton’) alone. When the latter was injected combined with either macrotriton or colloidal gold particles, autophagy increased to a level observed with Triton WR 1339 and beyond the levels determined for either component alone. Thus, autophagy appears to be stimulated by endocytosis. Autolysosomes are transformed within a few hours to peribiliary ‘dense bodies’. These do not display any significant turnover rate within a period of several days; therefore, dense bodies could be prelabelled with colloidal gold in order to show that they are the target organelles ingesting (macro-)Triton. According to difference spectra autophagy leads to a considerable concentration of microsomal and mitochondria! cytochromes in (macro-)Triton-filled lysosomes far beyond the level detected in ‘normal’ lysosomes. Because of the participation of both hetero- and autophagy in the formation of Triton WR 1339-filled lysosomes they have to be classified as telolysosomes.