Autophagy revisited: a conversation with Christian de Duve

The word "autophagy" was invented by Christian de Duve, the discoverer of lysosomes, who also initiated the first experiments that provided clear biochemical proof of the involvement of lysosomes in this process. I recently had an opportunity to speak with de Duve and some of his former coworkers about their reminiscences of these events, and am pleased to share what they had to say with the readers of this journal, many of whom may not be familiar with this historical background to their field of interest.     

Heterogeneity of lysosomes in human fibroblasts

Summary Lysosomes are defined traditionally with the marker enzyme acid phosphatase. We showed recently that lysosomes from human fibroblasts can be separated into a light and dense fraction as well as prelysosomal population. We now provide evidence that although acid phosphatase is enriched in all three fractions, the marker enzyme in the prelysosomal compartment is qualitatively distinct from that of the lysosomes. Ultrastructural analysis showed that the acid phosphatase in the prelysosomal vesicles deposited an extremely electron-dense reaction product, entirely obliterating the lumen of the vesicle, in contrast to that of the light and dense lysosomes which deposited a fine and diffuse product scattered throughout the luminal space. Biochemical analysis showed that only 51% of the acid phosphatase in the prelysosomes was inhibited by tartrate, while 80% of that in the lysosomes was tartrate-inhibitable. Immunoprecipitation with antibodies specific for various isozymes of acid phosphatase showed that 39% of the acid phosphatase in the prelysosomes was of the lysosomal type whereas over 5007o of the acid phosphatase in the lysosomes was of this type. These results showed that acid phosphatase in the prelysosomes of human cultured fibroblasts can be distinguished from that of the lysosomes cytochemically, biochemically, and immunologically and that lysosomes, as marked by acid phosphatase, are a heterogeneous organelle.     

Integumentary resorption and collagen synthesis during regression of headless pedicellariae in Sphaerechinus granularis (Echinodermata: Echinoidea)

Ultrastructure of the resorption of integumentary tissues (ligaments, muscles, fibrous tissue, nerves, and skeleton) and the synthesis of collagen is described for the first time in echinoderms. Resorption is cell-mediated. Phagocytic cells are characterized by Golgi-derived putative primary lysosomes. Numerous secondary lysosomes and residual bodies occur in the bodies and processes of phagocytic cells. They engulf whole muscle cells and nerve fibres, as well as collagen fibril segments that exceed 1.5 m in length. Skeletoclastic cells resemble vertebrate osteoclasts, showing a ruffled border, lysosomes, and numerous mitochondria. They surround trabeculae with thick processes to delimit a tubular resorption site. Collagen synthesis occurs in the space formerly occupied by resorbed tissues. Synthesis is performed by fibroblastic cells containing organelles typical of vertebrate fibroblasts, namely distended cisternae of rough endoplasmic reticulum, Golgi cisternae with distended edges, and procollagen granules. Procollagen granules are apparently exocytosed directly to the extracellular matrix. Evidence indicates that resorbing (phagocytic and skeletoclastic) cells and fibroblastic cells may belong to a common phagocyte lineage. These cells share the ability to form elaborate processes and to become syncytial, and their nuclei exhibit iron-containing crystals.This project was supported by Contract 2.4527.89 from the Fonds de la Recherche Fondamentale Collective (Belgium). P.D. is a Research Associate of the National Fund for Scientific Research (Belgium). Contribution of the Centre Interuniversitaire de Biologie Marine.     

In vivo endocytosis by bristle coated pits of protein tracers and their intracellular transport in the endothelial cells lining the sinuses of the liver. II. The endosomal-lysosomal transformation

The events leading to lysosomal activity in the sinus endothelium of the rat liver have been studied by means of intravascularly injected ferritin at time intervals ranging from 0.5 min to 1 hr after administration. From 6 min on, the dense body-type lysosomes contain ferritin. There are direct luminal communications of transfer tubules with these lysosomes. In time, there is a marked progressive increase in the number of ferritin-containing dense body-type lysosomes. No formation of lysosomes de novo nor a direct fusion of endosomes with lysosomes has been observed. Endosomes, however, continue to be formed as endocytosis continues. These observations are interpreted as indicating a transport of hydrolytic enzymes by the transfer tubules to the newly formed ferritin containing endosomes, which in this way are transformed into ferritin containing lysosomes. The ferritin-containing lysosomes increase considerably in size by fusing with each other. Continued endocytosis of ferritin leads to an increase of ferritin density in the dense bodies. This increase in particle density cannot be explained solely on the basis of transport by luminal fusion of the endocytic organelles, but requires an active transport mechanism. Administration of low doses of ferritin shows that the bristle coated pits of the sinus endothelium have a high degree of in vivo affinity for protein and that this endothelium must be considered to be an avid catabolic endocytic system.     

Lysosomes of root tip cells in corn seedlings

Summary Nine acid hydrolases are present in lysosomes which are found in the mitochondrial fraction of a cell-free extract prepared from root tips of corn seedlings.Light and heavy lysosomes can be distinguished. The latter are sedimentable in a sucrose-medium, the former only in sorbitol-medium. The fraction of heavy lysosomes is in turn composed of at least three populations of lysosomes differing in density and enzyme content.Light lysosomes are membrane-bound particles with diameters from 0.3 to 1.5 . Electron micrographs of frozen-etched tissue and isolated particles provide evidence that light lysosomes are identical with small vacuoles. This type of lysosome is characterized by presence of transaminases in addition to that of hydrolases. Heavy lysosomes are small spheres (diameters from 0.1–0.3 ) with membranes resembling those of vacuoles and of the endoplasmic reticulum. These lysosomes are characterized by high specific activities of two oxydoreductases known to occur also in the membranes of the reticulum.The different types of particles are thought to represent stages of the development of the lysosomal apparatus; according to this hypothesis the large vacuole of parenchymatous cells represents the end product of this process.     

Age-related decline in chaperone-mediated autophagy

Intracellular protein degradation rates decrease with age in many tissues and organs. In cultured cells, chaperone-mediated autophagy, which is responsible for the selective degradation of cytosolic proteins in lysosomes, decreases with age. In this work we use lysosomes isolated from rat liver to analyze age-related changes in the levels and activities of the main components of chaperone-mediated autophagy. Lysosomes from "old" (22-month-old) rats show lower rates of chaperone-mediated autophagy, and both substrate binding to the lysosomal membrane and transport into lysosomes decline with age. A progressive age-related decrease in the levels of the lysosome-associated membrane protein type 2a that acts as a receptor for chaperone-mediated autophagy was responsible for decreased substrate binding in lysosomes from old rats as well as from late passage human fibroblasts. The cytosolic levels and activity of the 73-kDa heat-shock cognate protein required for substrate targeting to lysosomes were unchanged with age. The levels of lysosome-associated hsc73 were increased only in the oldest rats. This increase may be an attempt to compensate for reduced activity of the pathway with age.     

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.     

„Cellules de thyroïdectomie“ et „cellules de castration“ chez la Caille japonaise,Coturnix coturnix japonica

Résumé On compare l'ultrastructure et la localisation des phosphatases acides au niveau des cellules hypophysaires delta et beta, chez des Cailles mâles thyroïdectomisées et maintenues en photopériode courte ou bien castrées, puis placées en photopériode longue. On étudie en outre, dans ces deux cas, les effets d'injections de doses croissantes de thyroxine.La thyroïdectomie provoque la transformation des cellules delta en cellules de thyroïdectomie groupées en îlots à la périphérie du lobe céphalique. Ces cellules sont pauvres en phosphatases acides. La thyroxine (10 g/j pendant 2 jours) provoque la régression de ces cellules et l'apparition de lysosomes. Les cellules beta ne sont pas modifiées par la thyroïdectomie.La castration-photostimulation stimule les cellules beta localisées dans le lobe céphalique. Elle provoque dans les deux lobes de la glande l'hypertrophie et la vacuolisation des cellules delta qui se distinguent des cellules de thyroïdectomie par la présence de nombreux lysosomes. La thyroxine freine simultanément l'activation des cellules delta et des cellules beta, en provoquant la formation de lysosomes, mais la dose efficace chez le mâle photostimulé (20 g et 60 g/j pendant 5 jours) est sans effet chez le castrat photostimulé (dose efficace 180 g/j).Pour interpréter ces faits, on admet que les cellules delta, thyréotropes et les cellules beta, gonadotropes, seraient simultanément soumises à un contrôle freinateur des hormones thyroïdiennes et des stéroïdes mâles.

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Thyroidectomy cells and castration cells in the Japanese quail, Coturnix coturnix japonica Ultrastructure and cytoenzymology

Summary The ultrastructure and the localization of acid-phosphatase activity are compared in beta and delta pituitary cells of male Japanese quail, either thyroidectomized and maintained in short days, or castrated then put in long days. Moreover, in these two cases, the effects of brief treatments with increasing doses of thyroxine are studied.Thyroidectomy induces transformation of delta cells into thyroidectomy cells arranged in clumps at the periphery of the cephalic lobe. The acid-phosphatase activity of such cells is low. Thyroxine (10 g per day for two days) causes regression of these cells and the appearance of numerous lysosomes. Beta cells are not modified by thyroidectomy.Castration and exposure to long days stimulate beta cells, localized in the cephalic lobe. It induces, also, in both pituitary lobes, hypertrophy and vacuolization of delta cells which differ from thyroidectomy cells by the presence of numerous lysosomes.Thyroxine in photostimulated quail inhibits both delta- and beta-cell stimulation and increases the frequency of lysosomes but the effective doses on males (20 g or 60 g per day for five days) are inactive on castrates, the response of which is obtained with 180 g per day.In order to explain these data, a hypothesis is suggested: Thyrotropic delta cells and gonadotropic beta cells are both subject to a double inhibiting control by thyroid hormones and male steroids.

Nous remercions très vivement pour leur excellente collaboration technique Mme Renée Picart (préparation des tissus pour la microscopie électronique) et M. Claude Pennarun (photographe).     

Dynein-dependent movement of autophagosomes mediates efficient encounters with lysosomes

Autophagy is a membrane trafficking pathway that carries cytosolic components to the lysosome for degradation. During this process, the autophagosome, a double-membraned organelle, is generated de novo, sequesters cytoplasmic proteins and organelles, and delivers them to lysosomes. However, the mechanism by which autophagosomes are targeted to lysosomes has not been determined. Here, we observed the real-time behavior of microtubule-associated protein light chain 3 (LC3), which localizes to autophagosomes, and showed that autophagosomes move in a microtubule- and dynein-dynactin motor complex-dependent manner. After formation, autophagosomes show a rapid vectorial movement in the direction of the centrosome, where lysosomes are usually concentrated. Microinjection of antibodies against LC3 inhibited this movement; furthermore, using FRAP, we showed that anti-LC3 antibody injection caused a defect in targeting of autophagosomes to lysosomes. Collectively, our data demonstrate the functional significance of autophagosome movement that enables effective delivery from the cytosol to lysosomes.     

Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient

A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al, (1955, Biochem. J. 60: 604-617), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction if set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10--12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane. The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.     

Effects of leupeptin on endocytosis and membrane recycling in rat visceral yolk-sac endoderm

Summary The effect of exposure to leupeptin (25 g/ml for 24 h) on the endocytotic activity and the membrane flow of apical cell membranes was studied in endodermal cells of cultured rat visceral yolk sacs by applying a doublelabelling method using concanavalin-A ferritin (Con-A Fer) and horseradish peroxidase (HRP). Control and leupeptintreated yolk sacs were labelled with Con-A Fer at 4°C and then incubated with HRP for 5, 15 or 60 min at 37°C. In controls, HRP reaction product was detected after 5 min in many of the apical vacuoles as well as a few lysosomes; after 15 min, reaction product was observed in all apical vacuoles and in lysosomes of various sizes. These HRP-positive structures usually contained a variable amount of membrane-bound Fer. After 60 min, all apical vacuoles and almost all lysosomes exhibited HRP reactions, but only some of these structures contained Fer particles. At this time, many apical canaliculi (which are involved in membrane recycling) exhibited positive HRP reactions and sometimes also contained Fer particles. In leupeptin-treated cells, HRP reaction product and variable amounts of membrane-bound Fer particles were found in apical vacuoles after 5 min; after 15 min, both labels were also observed in some small lysosomes, and after 60 min, they were found in all apical vacuoles as well as some small and middle-sized lysosomes. Significantly fewer labelled apical vacuoles, lysosomes and apical canaliculi were present after leupeptin treatment than in controls at corresponding times. At all times examined, the giant lysosomes found in leupeptintreated cells did not exhibit any labelling. These findings indicate that, after leupeptin treatment, both endocytotic activity and membrane recycling decrease, and that fusions of the apical vacuolar system with giant lysosomes are retarded or inhibited.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

The localisation of acid phosphatase activity changes in lysosomes in the adrenal zona fasciculata of intact and hypophysectomized rats following ACTH administration

Summary In the cells of the zona fasciculata of intact adult rats lysosomes can be found in medium amounts, measuring 0.2–0.4 in diameter. After treatment with ACTH, the lysosomes in the zona fasciculata of the intact animals increase in number and size, and lysosome-lipid conglomerates are formed. In hypophysectomized animals, the lysosomes in the zona fasciculata decrease in number and size in proportion to the time elapsed. Treatment with ACTH given 2 to 4 weeks after hypophysectomy, resulted in the increase in number and size of the lysosomes. Under such circumstances lysosomes appear in conspicuously large numbers around lipid droplets, which frequently coalesce with them. The possible role of lysosomes is discussed.     

Fine structural localization of acid and alkaline phosphatase activities in the absorbing cells of the duodenum of rodents

Summary The morphology of the absorbing cells of the duodenal villi in the mouse, the rat, the hamster and the guinea-pig is described. The polymorphism of the dense bodies is pointed out. The fine localization of acid and alkaline phosphatase is investigated and compared. In all the species, acid phosphatase activity is observed in the dense bodies, Golgi vesicles and rare smooth endoplasmic profiles. Alkaline phosphatase is localized on the microvilli, Golgi apparatus, some smooth endoplasmic cisternae and numerous dense bodies. The presence of an alkaline phosphatase reaction in the dense bodies, probably lysosomes, of the absorbing cells is discussed. It is assumed that this enzyme follows a catabolic pathway and is finally degraded in the lysosomes.Abbreviations used AlPase alkaline phosphatase - AcPase acid phosphatase This work was done thanks to the contract C.E.N./A.I.E.A. N 347/RB and thanks to grants from the Fonds de la Recherche scientifique fondamentale collective.     

Ultrastructural and immunocytochemical study of skin fibroblasts from normal and sialidosis patients

The objectives of this study were to analyze morphologically, morphometrically and immunocytochemically the lysosomal compartment of normal fibroblasts and of fibroblasts with neuraminidase deficiency. The immunocytochemical analyses consisted of quantifying the distribution of saposins and -galactosidase in the lysosomes of these cells to test the hypothesis that neuraminisdase deficiency is associated with an impairment in the transport of these proteins to the lysosomal compartment. To test this idea, cultured skin fibroblasts of patients with or without sialidosis were prepared for electron microscopy and probed with antibodies against lysosomal -galactosidase and lysosomal saposins. The lysosomes of the affected cells had an abnormal accumulation of incompletely digested membranes which was associated with a significant lowering in the density of antigenic sites per lysosome. However, due to a significant increase in the number of lysosomes per affected cell, the total number of antigenic sites in control and neuraminidase deficient cells was similar. This presumably compensatory effect indicates that although the rate of production of -galactosidase and saposins remains unchanged, the transport of these molecules to the lysosomes is somehow affected. Our data also indicate that in the fibroblasts, lysosomes require a normal concentration of the three enzymes to maintain neuraminidase activity and sphingolipid degradation.     

On the histochemical demonstration of N-acetyl-β-galactosaminidase

Summary Aging neurons accumulate lipofuscin pigment granules which appear to be secondary lysosomes of the residual body variety. The biological significance of the residual bodies is debated. They were here studied with the aim of testing a hypothesis that the membranes surrounding these granules might be more vulnerable than the membranes around younger types of lysosomes.For this purpose large motor neurons of young and old rats were compared with respect to lysosomal membrane latency, using a modified Bitensky lysosomal lability test. Utilizing successively increasing incubation times, the lysosomes of old neurons, in particular the residual bodies in polar aggregates of old neurons—presumed to represent lipofuscin pigment granules—were found to have a clearly reduced latency in comparison with lysosomes of young neurons.These findings support the notion that the residual bodies are more fragile than younger lysosomes.Supported by the Swedish Medical Research Council (Project No. 12X-2037).     

Endocytic delivery to lysosomes mediated by concurrent fusion and kissing events in living cells

In mammalian cells, macromolecules internalized by endocytosis are transported via endosomes for digestion by lysosomal acid hydrolases . The mechanism by which endosomes and lysosomes exchange content remains equivocal . However, lysosomes are reusable organelles because they remain accessible to endocytic enzyme replacement therapies and undergo content mixing with late endosomes . The maturation model, which proposes that endosomes mature into lysosomes , cannot explain these observations. Three mechanisms for content mixing have been proposed. The first is vesicular transport, best supported by a yeast cell-free assay . The second suggests that endosomes and lysosomes engage in repeated transient fusions termed "kiss-and-run" . The third is that endosomes and lysosomes fuse completely, yielding hybrid compartments from which lysosomes reform , termed "fusion-fission" . We utilized time-lapse confocal microscopy to test these hypotheses in living cells. Lysosomes were loaded with rhodamine dextran by pulse-chase, and subsequently late endosomes were loaded with Oregon green 488 dextran. Direct fusions were observed between endosomes and lysosomes, and one such event was captured by correlative electron microscopy. Fluorescence intensity analyses of endosomes that encountered lysosomes revealed a gradual accumulation of lysosomal content. Our data are compatible with a requirement for direct contact between organelles before content is exchanged.     

X-ray microanalysis of colloidal-gold-labelled lysosomes in rat liver sinusoidal cells after incubation for acid phosphatase activity

Summary The lysosomal apparatus of the Kupffer and endothelial cells of the sinusoidal lining of the rat liver was found to take up colloidal-gold particles with a mean diameter of 5 nm, prepared according to a modified method. After incubation of the glutaraldehyde-perfusion-fixed tissue in a lead-containing medium for the demonstration of acid phosphatase activity, a reaction product was observed in the gold-loaded lysosomes. By X-ray microanalysis of such lysosomes, the presence of osmium, gold and lead was detected qualitatively in the unstained sections from the tissue, which after the incubation had been post-fixed with an OsO₄-solution to which K₄Fe(CN)₆ had been added to enhance the contrast. The quantitative computer-assisted processing of the X-ray microanalytical data from such lysosomes enabled to determine the gold-to-lead ratio and the individual gold and lead peak intensities derived from both the M and L values in the spectra. On the basis of these results and those obtained similarly in control lysosomes containing either only gold or only lead phosphate precipitate, it was found that only the L values were reliable, whereas the M values from the same lysosomal spectra were unrealistic, due to deconvolution problems in the computer programs applied. Based upon the L values it was found that among the population of lysosomes in single Kupffer cells, studied after a 60-min interval between the injection of the gold colloid and fixation, three types of lysosomal contents could be quantitated by X-ray microanalysis, viz. one type with only gold, one with only lead, one with gold and lead, in various ratios. This quantitative approach might make it possible to detect variations in lysosomal composition associated with ageing.The unit for analytical electron microscopy was established by collaboration between: the Erasmus University of Rotterdam (W.C. de Bruijn), the University of Leiden, and the Organization for Health Research TNO. The analytical electron microscope was purchased with funds from the Netherlands Organization for Pure Scientific Research (ZWO)     

Ultrastructure of STH cells of the pars distalis of hepatectomized mice

Summary An electron microscopic analysis was performed on the pars distalis of the hypophysis of hepatectomized mice. Intact and sham operated mice served as controls. The STH cells presented striking changes that were most intense and widespread in those animals sacrificed at midnight of the second day after hepatectomy. These changes can be summarized as follows: 1) Hypertrophy of the Golgi complex with increased number of immature granules within the Golgi zone. This change appeared also in otherwise unmodified STH cells. 2) Strong dilatation of the endoplasmic reticulum whose cisternae contained much electron dense material. 3) Granules with partially diminished electron density, some of them in spatial relation with the plasma membrane and others swelling and bursting within the cytoplasm. All transitions between unchanged 350–400 m granules and extremely altered ones, were seen. 4) Release sites, characterized by dense zones in the plasmalemma, close to aggregates of electron lucent microvesicles, and almost empty granule membranes. 5) Increase in the density of the mitochondria which appeared grouped near the Golgi zone. 6) Increase in the number of large lysosomes of the autophagic vacuole type. 7) Irregular nuclear outlines. These data suggest increased synthesis and release of growth hormone in STH cells stimulated by hepatectomy.Work carried out with the financial assistance of a grant of the Comision de Investigation cientifica de la Universidad Nacional de La Plata. Thanks are due to the members of the technical staff of the Institute for their technical assistance.     

Responsive microtubule dynamics promote cell invasion by Trypanosoma cruzi

The American trypanosome, Trypanosoma cruzi, can invade non-phagocytic cell types by a G-protein-mediated, calcium-dependent mechanism, in which the cell's natural puncture repair mechanism is usurped in order to recruit lysosomes to the parasite/host cell junction or 'parasite synapse.' The fusion of lysosomes necessary for construction of the nascent parasitophorous vacuole is achieved by directed trafficking along microtubules. We demonstrate altered host cell microtubule dynamics during the initial stages of the entry process involving de novo microtubule polymerization from the cytoplasmic face of the parasite synapse which appears to serve as a secondary microtubule organizing centre. The net result of these dynamic changes to the host cell's microtubule cytoskeleton is the development of the necessary infrastructure for transport of lysosomes to the parasite synapse.