Studies on the mechanisms of autophagy: maturation of the autophagic vacuole

Data presented in the accompanying paper suggests nascent autophagic vacuoles are formed from RER (Dunn, W. A. 1990. J. Cell Biol. 110:1923-1933). In the present report, the maturation of newly formed or nascent autophagic vacuoles into degradative vacuoles was examined using morphological and biochemical methods combined with immunological probes. Within 15 min of formation, autophagic vacuoles acquired acid hydrolases and lysosomal membrane proteins, thus becoming degradative vacuoles. A previously undescribed type of autophagic vacuole was also identified having characteristics of both nascent and degradative vacuoles, but was different from lysosomes. This intermediate compartment contained only small amounts of cathepsin L in comparison to lysosomes and was bound by a double membrane, typical of nascent vacuoles. However, unlike nascent vacuoles vet comparable to degradative vacuoles, these vacuoles were acidic and contained the lysosomal membrane protein, lgp120, at the outer limiting membrane. The results were consistent with the stepwise acquisition of lysosomal membrane proteins and hydrolases. The presence of mannose-6-phosphate receptor in autophagic vacuoles suggested a possible role of this receptor in the delivery of newly synthesized hydrolases from the Golgi apparatus. However, tunicamycin had no significant effect on the amount of mature acid hydrolases present in a preparation of autophagic vacuoles isolated from a metrizamide gradient. Combined, the results suggested nascent autophagic vacuoles mature into degradative vacuoles in a stepwise fashion: (a) acquisition of lysosomal membrane proteins by fusing with a vesicle deficient in hydrolytic enzymes (e.g., prelysosome); (b) vacuole acidification; and (c) acquisition of hydrolases by fusing with preexisting lysosomes or Golgi apparatus-derived vesicles.     

红豆草根瘤侵染细胞中液泡内含物的超微结构特征

用透射电镜研究了红豆草（Onobrychis viciifolia）根瘤侵染细胞中液泡内含物的超微结构特征。结果表明,早期发育侵染细胞的液泡中只含有少量的纤维状物质。随着细胞的发育,液泡不断变大,液泡中的纤维状物质和膜状物质越来越多。在中央液泡形成后,液泡中的纤维状物质逐渐减少,类细胞质、泡状和膜状物质明显增多,它们常由一层来自液泡膜的膜包围,其形状一般近似圆形或椭圆形。液泡内含物的大量出现可能与红豆草及其根瘤具有高度的抗旱件有关。     

Studies on the mechanisms of autophagy: formation of the autophagic vacuole

Autophagic vacuoles form within 15 min of perfusing a liver with amino acid-depleted medium. These vacuoles are bound by a "smooth" double membrane and do not contain acid phosphatase activity. In an attempt to identify the membrane source of these vacuoles, I have used morphological techniques combined with immunological probes to localize specific membrane antigens to the limiting membranes of newly formed or nascent autophagic vacuoles. Antibodies to three integral membrane proteins of the plasma membrane (CE9, HA4, and epidermal growth factor receptor) and one of the Golgi apparatus (sialyltransferase) did not label these vacuoles. Internalized epidermal growth factor and its membrane receptor were not found in nascent autophagic vacuoles but were present in lysosome-like degradative autophagic vacuoles. All these results suggested that autophagic vacuoles were not formed from plasma membrane, Golgi apparatus, or endosome constituents. Antisera prepared against integral membrane proteins (14, 25, and 40 kD) of the RER was found to label the inner and outer limiting membranes of almost all nascent autophagic vacuoles. In addition, ribophorin II was identified at the limiting membranes of many nascent autophagic vacuoles. Finally, secretory proteins, rat serum albumin and alpha 2u- globulin, were localized to the lumen of the RER and to the intramembrane space between the inner and outer membranes of some of these vacuoles. The results were consistent with the formation of autophagic vacuoles from ribosome-free regions of the RER.     

Food Vacuoles During Heat-Shock-Induced Transformation from the Microstomal to the Macrostomal Cell Type of Tetrahymena vorax1

ABSTRACT. The heat-shock method for induction of the macrostomal form of Tetrahymena vorax involves the transfer of cells to reduced nutrient medium and the application of a series of elevated temperature shocks followed by washing the protozoa into inorganic medium. The component of the procedure that had the greatest effect on food vacuoles was the heat shocks. At the end of the heat shocks, cells formed vacuoles at a lower rate than non-heat-shocked cells, but the size of the vacuoles formed was larger and the protozoa contained an increased number of vacuoles and total vacuolar membrane. The rate was further reduced by washing cells into nonnutrient medium. In the absence of the heat shocks, the medium had little effect on the capacity of the cells to form vacuoles although after 7.5 h in inorganic medium, the vacuoles formed were smaller and the protozoa possessed fewer vacuoles and therefore less vacuolar membrane. The amount of membrane required to form the cytopharyngeal pouch of the macrostomal cell type was equivalent to the surface area of food vacuoles present in cells prior to the onset of the heat shocks, but the number and surface area of vacuoles decline between the time of oral resorption and pouch development.     

Invaginated apical vacuoles in the cells of the proximal convoluted tubule in the rat kidney

Summary Following perfusion fixation of the rat kidney with glutaraldehyde the proximal tubule cells display small apical vacuoles, large apical vacuoles, and apical vacuoles in which a part of the limiting membrane is invaginated into the vacuole. These invaginated apical vacuoles occur more frequently in proximal convoluted tubules than in proximal straight tubules. One tubular cell may contain apical vacuoles of different sizes and stages of invagination, ranging from larger vacuoles with a wide lumen and a small area of invaginated membrane to smaller elements with no apparent lumen and a large area of invaginated membrane. Invaginated apical vacuoles lie either singly in the cytoplasm or close to the membranes of other apical vacuoles, but never in contact with the cell membrane or the membranes of lysosomes, endoplasmic reticulum, Golgi apparatus, mitochondria and peroxisomes.These findings suggest that the invaginated apical vacuoles are not fixation artifacts, but rather develop in living state in cells of the proximal tubule from spherical endocytotic elements.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Fine structural and ultracytochemical studies on the lymphocytes in three types of genetic mucopolysaccharidoses

Lymphocytes from 6 patients with 3 types of genetic mucopolysaccharidoses (Hurler's syndrome, Hunter's syndrome and Morquio's syndrome) contained numberous vacuoles in their cytoplasm. The size of the vacuoles ranged from approximately 300 nm to 750 nm. The percentage of the lymphocytes with vacuoles varied from 10% to 38%. The vacuoles showed acid phosphatase activity, which indicated their lysosommal nature. Staining with dialyzed iron solution usually localized acid mucosubstance in the peripheral region of these vacuoles after glutaraldehyde fixation. Ferritin and horseradish peroxidase were observed in the vacuoles after incubation of the patient's lymphocytes with these tracers. This finding indicates the participation of endocytosis in the formation of these vacuoles.     

Ultrastructural localization of acid protein polysaccharides and calcium in the vacuoles of the chicken thrombocyte.

The coexistence of acid protein polysaccharides and calcium in the vacuoles of chicken thrombocytes were studied by means of ultrahistochemical methods and electron probe X-ray microanalysis. The thrombocytes possessed large vacuoles of a surface connected membrane system. After both ruthenium red staining and tannic acid fixation the innersurface coat of vacuoles was always strongly and continously visualized. Electron microscopic X-ray microprobe analysis of antimonate precipitates in thrombocytes fixed in K-antimonate-OsO4 revealed calcium localization on the innersurface of vacuoles. From these facts it seems likely that the vacuoles of the surface connected membrane system may take part in the pool or the transport of calcium.     

Vacuolation: Origin and development of the lysosomal apparatus in root-tip cells

Summary The morphology of vacuolation has been investigated in root tip cells of corn using the freeze-etching technique. The genesis of vacuoles involves the following processes: a) Formation of small, endoplasmic-reticulum (ER)-derived vesicles (provacuoles); b) fusion of provacuoles resulting in the formation of small vacuoles, and followed by fusion and expansion of vacuoles; c) incorporation of large, dictyosome-derived vesicles into vacuoles by invagination of the tonoplast; d) invagination of the tonoplast resulting in the incorporation of cytoplasmic material into vacuoles. The morphological findings are correlated with biochemical data obtained from isolated vacuoles (lysosomes). Provacuoles (ER-derived vesicles) are shown to be primary lysosomes; their hydrolases arise from the ER. Vacuoles represent secondary lysosomes (digestive vacuoles) of the higher-plant cell. The metabolic role of lytic processes proceeding in the lysosomal apparatus is discussed.     

Intramembrane particles and filipin labelling on the membranes of autophagic vacuoles and lysosomes in mouse liver

Summary Morphologically detectable protein (intramembrane particles) and cholesterol (filipin labelling) in the membranes of autophagic vacuoles and lysosomes were studied in mouse hepatocytes using thin-section and freeze-fracture electron microscopy. Both isolated autophagic vacuoles and lysosomes, and intact tissue blocks were used due to the facts (i) that lysosomes are difficult to recognize in freeze-fracture replicas of intact hepatocytes, and (i) that filipin penetration into the tissue blocks is unsatisfactory. Intramembrane particle density was low in the membranes of early autophagic vacuoles (defined as round-shaped vacuoles in which an inner membrane parallel with the outer limiting membrane was clearly visible). The lysosomal membranes contained considerably more intramembrane particles. Particle-rich lysosomes or other vesicles were observed to fuse with the early autophagic vacuoles. The membranes of nascent autophagic vacuoles with morphologically intact contents were usually not labelled by filipin, whereas the membranes of all other autophagic vacuoles and lysosomes were heavily labelled. The increased cholesterol in the membranes of slightly older autophagic vacuoles is presumably derived from cholesterol-rich lysosomes or other vesicles fusing with the vacuoles and from the degrading organelles inside the autophagic vacuoles.     

大鼠垂体细胞液泡内存在黄体生成激素（LH）

研究者普遍认为糖蛋白激素存在于促性腺激素（gonadotrophin,GTH)细胞的颗粒内,目前在生殖内分泌领域内对糖蛋白激素形成与释放的研究也主要集中在细胞内颗粒的变化上,我们近年的研究发现,大鼠垂体GTH细胞内黄体生成激素（luteinizing hormone,LH)的分泌与细胞内液泡的形态变化有密切的关系。铁形态也随液泡的形态变化而变化,因而推测“LH的储存与释放可能与液泡有极大的关系”,为进一步揭示垂体细胞的液泡内是否存在LH和探讨哺乳动物垂体细胞的液泡是否具有储存与释放LH的功能,本研究对大鼠垂体细胞的液泡进行了分离和纯化。用SDS－PAGE,Western immunobloting及Con A/HRP等方法分别对纯化的垂体,大脑皮层及肝脏组织的液泡进行了蛋白质,LH及糖蛋白的分析。结果显示：（1）垂体,皮层及肝脏细胞的液泡内均含有丰富的,分子量大小不等的蛋白质成分,不同组织的细胞液泡内蛋白质成分有许多是相似的;（2）垂体组织及其液泡内均含有LH,而且在相同浓度的蛋白量中,两者LH的水平并无明显差异;（3）垂体,皮层和肝脏组织液泡内均有分子量不同的糖蛋白,但只有垂体细胞的液泡内才有与LH位置相同的糖蛋白染色谱带。上述结果表明：虽然哺乳动物不同组织的细胞液泡内含有许多相似的蛋白质成分,但LH是特异性地存在于垂体细胞液泡内。在这些LH分子中,至少有一部分是已经装配了糖基的完整LH分子。因此,垂体细胞的液泡有可能具有储存与释放LH的功能。     

Filipin labelling and intramembrane particles on the membranes of early and later autophagic vacuoles in Ehrlich ascites cells

Cholesterol and intramembrane particle distribution on autophagic vacuole membranes was studied in Ehrlich ascites cells using filipin labelling and freeze-fracture electron microscopy. Unsaturated fatty acids were stained using imidazole-buffered osmium tetroxide. Autophagocytosis was induced with vinblastine, and early autophagic vacuoles were accumulated by lowering the ATP level in the cells with iodoacetate. Filipin labelling was observed in the limiting membranes of later, apparently hydrolase-containing autophagic vacuoles, whereas the most newly-formed, double-membrane limited vacuoles were not labelled. The limiting membranes of late, residual body-type vacuoles either showed patchy filipin-induced deformation or were completely smooth. Imidazole-buffered osmium tetroxide stained the membranes of newly-formed or developing autophagic vacuoles partly or entirely. The membranes of older vacuoles stained more weakly. Intramembrane particle density on the P-face of the outer limiting membranes of newly-formed autophagic vacuoles was similar to that on endoplasmic reticulum, and the density seemed to increase slightly later on. The size of the P-face particles increased when the vacuoles became older. The limiting membranes of late, residual body-type vacuoles were almost smooth. The inner limiting membranes and the membranes inside the autophagic were always almost particle-free. In conclusion, the amount of cholesterol, unsaturated fatty acids and protein in autophagic vacuole membranes changes during vacuole maturation.     

Ultrastructural and cytochemical studies on the developing adhesive disc of Boston Ivy tendrils

Summary Ultrastructural observations of the immature adhesive disc from tendrils of Boston Ivy showed that the peripheral cells, which are the presumptive contact layer, contain vacuoles of varied sizes which are filled with electron-dense aggregates. In small vacuoles, these deposits were appressed to the tonoplast and fusion of these small vacuoles with the large vacuoles apparently occurs. Cells from the central zone were largely parenchymatous. The vacuoles of many of these parenchyma cells contained electron-dense spheres and hemispheres of material either appressed to the tonoplast or within the vacuole lumen. In these cells, the vacuole-cytoplasm interface was characterized by a filiform network of interconnected membranes. Positive reactions with reagents for the identification of polyphenols indicate that the vacuoles of nearly all the peripheral cells and scattered cells of the central zone contain tanniniferous substances. Insoluble carbohydrates also occur in the vacuoles of the peripheral cells, but they contain little or no protein or lipid.     

Origin and formation of different types of vacuoles induced by the multiplication of the alphavirus Sindbis virus in various cell systems

Spherule-containing vacuoles and nucleocapsid-bearing vacuoles (cytopathic vacuoles types 1 and 2 respectively of Grimley et al. 1968) induced by Alphavirus Sindbis were studied in brains from newborn mice, chicken embryo fibroblasts, and two lines of tumoral glial cells from muridae. Endoplasmic reticulum (ER) elements and finely granular electron-dense material also seen in contact with nucleocapsids seemed to be involved in the formation of the classical single-membrane spherule-containing vacuoles. A second type of spherule-containing vacuoles were characterized by their double membrane and an amorphous electron-dense content and were probably derived from mitochondria. Nucleocapsid-bearing vacuoles were formed from modified ER elements and seemed to be linked to excessive synthesis of viral material. Such ER alterations were not observed in RG6 cells. In these cells, there were only spherule-containing vacuoles, while nucleocapsids were seen associated with the cytoplasmic membrane only.     

A procedure for isolating vacuoles from leaves of the halophyte Suaeda maritima

Abstract. A method is described here for isolating protoplasts and vacuoles from leaves of the halophyte Suacda maritima. Integrity of the protoplasts and vacuoles was tested by staining and shown to be more than 75%, while use of biochemical markers, staining and light microscopy suggested a high degree of purity of the vacuoles. Phosphatase and NADH cytochrome- c -reductase were associated with vacuoles; phosphatase showed an eight-fold enrichment and NADH cytochrome- c -reductase a 3.5-fold enrichment relative to protoplasts. The vacuoles contained only 15% of the protein in protoplasts.     

Ergosterol is required for the Sec18/ATP-dependent priming step of homotypic vacuole fusion

In vitro homotypic fusion of yeast vacuoles occurs in three stages: priming, the Sec18 (NSF)-mediated changes that precede vacuole association; docking, the Ypt7 and SNARE-mediated pairing of vacuoles; and fusion, mediated by calmodulin/V0/t-SNARE interactions. Defects in catalysts of each stage result in fragmented (unfused) vacuoles. Strains with deletions in any of ERG genes 3-6, lacking normal ergosterol biosynthesis, have fragmented vacuoles. The ergosterol ligands filipin, nystatin and amphotericin B block the in vitro fusion of vacuoles from wild-type cells. Each of these inhibitors acts at the priming stage to inhibit Sec17p release from vacuoles. A reversible delay in Sec18p action prevents vacuoles from acquiring resistance to any of these three drugs, confirming that their action is on the normal fusion pathway. Ergosterol or cholesterol delivery to wild-type vacuoles stimulates their in vitro fusion, and the in vitro fusion of ergDelta vacuoles requires added sterol. The need for ergosterol for vacuole priming underscores the role of lipids in organizing the membrane elements of this complex reaction.     

Origin and function of the stalk-cell vacuole in Dictyostelium

Large vacuoles are characteristic of plant and fungal cells, and their origin has long attracted interest. The cellular slime mould provides a unique opportunity to study the de novo formation of vacuoles because, in its life cycle, a subset of the highly motile animal-like cells (prestalk cells) rapidly develops a single large vacuole and cellulosic cell wall to become plant-like cells (stalk cells). Here we describe the origin and process of vacuole formation using live-imaging of Dictyostelium cells expressing GFP-tagged ammonium transporter A (AmtA-GFP), which was found to reside on the membrane of stalk-cell vacuoles. We show that stalk-cell vacuoles originate from acidic vesicles and autophagosomes, which fuse to form autolysosomes. Their repeated fusion and expansion accompanied by concomitant cell wall formation enable the stalk cells to rapidly develop turgor pressure necessary to make the rigid stalk to hold the spores aloft. Contractile vacuoles, which are rich in H⁺-ATPase as in plant vacuoles, remained separate from these vacuoles. We further argue that AmtA may play an important role in the control of stalk-cell differentiation by modulating the pH of autolysosomes.     

Inactivation of the ppn1 gene exerts different effects on the metabolism of inorganic polyphosphates in the cytosol and the vacuoles of the yeast Saccharomyces cerevisiae

Inactivation of the PPN1 gene, encoding one of the enzymes involved in polyphosphate metabolism in the yeast Saccharomyces cerevisiae, was found to decrease exopolyphosphatase activity in the cytosol and vacuoles. This effect was more pronounced in the stationary growth phase than in the phase of active growth. The gene inactivation resulted in elimination of a approximately 440-kDa exopolyphosphatase in the vacuoles but did not influence a previously unknown vacuolar exopolyphosphatase with a molecular mass of >1000 kDa, which differed from the former enzyme in the requirement for bivalent cations and sensitivity to heparin. Inactivation of the PPN1 gene did not influence the level of polyphosphates in the cytosol but increased it more than twofold in the vacuoles. In this case, the polyphosphate chain length in the cytosol increased from 10-15 to 130 phosphate residues both in the stationary and active growth phases. In the vacuoles, the polyphosphate length increased only in the stationary growth phase. A conclusion can be made that the PPN1 gene product has different effects on polyphosphate metabolism in the cytosol and the vacuoles.     

Comparative analysis of the structure of the vacuolar system of the bladder granular cells in the frog and of the contractile vacuole complex of protozoans

A comparative morphological study of the vacuolar system of the frog urinary bladder epithelial cells and of the contractile vacuole complex of Paramecium caudatum enabled us to reveal some common structural elements in these: spongial channels and general vacuole reservoir. The structural similarity of these organoids seems to be the base of their analogous functions in the cell. Detection of vacuoles of various forms in different areas of granular cells may point to a possible migration of the vacuoles around their cytoplasm. Localization of spheric vacuoles in the innermost contact with the plasma membrane, and dilution of the intercellular space in this epithelial part may suggest an expulsion of the vacuole content in the basolateral part of the cell. The "contractile" vacuoles of granular cells are related to other intracellular structure: the Golgi apparatus, coated vesicles, microtubules, microfilaments.     

Development of an autophagic system in differentiating cells of the cellular slime moldDictyostelium discoideum

Summary Changes in an autophagic system during differentiation of cells ofDictyostelium discoideum, NC-4 were studied under light and electron microscopes, and it was demonstrated cytochemically that acid phosphatase was almost exclusively localized in food and autophagic vacuoles. Autophagic vacuoles first appeared during formation of loose aggregates, coupled with the defecation of food vacuoles. Autophagic vacuoles seem to originate from flat sacs which segregate parts of the cytoplasm. No acid phosphatase was detected in the vacuoles when first formed, but activity appeared later probably due to fusion with Golgi-like vesicles. When starved cells were not allowed to aggregate due to a low cell density, they formed no autophagic vacuoles but retained many food vacuoles. This indicates that the formation of autophagic vacuoles is not simply due to starvation, but to cell interaction mediated by cell contact. Autophagic vacuoles containing acid phosphatase rapidly increased in number in all cells in the early stage of aggregation. After papillae formed, however, they selectively decreased in the prespore cells, but developed further and grew larger in the prestalk cells.     

Electron microscopy study of deoxyribonuclease penetration into chick embryo fibroblasts and the effect of the enzyme on the cells

Peroxidase labeled DNase (0.25 g/ml) penetrated into the chick fibroblast culture cells by means of pinocytosis to be localized in pinocytotic vacuoles. Within a 24 hour-incubation of the cell culture with DNase a relative volume of vacuoles in cells increased from 4.6 to 19.5 per cent, the majority of pinocytic vacuoles being filled with labeled DNase. The incubation of cells with non-labeled DNase (24 hours) did not damage DNA-containing cell structures. The data obtained permit to explain the selective effect of the enzyme on the viral DNA and the inhibition of reproduction of DNA-containing viruses by the interaction of the viral DNA and DNase inside pinocytic vacuoles which protect the cells from the enzyme damaging effect.