Accumulation of autophagosomes after inhibition of hepatocytic protein degradation by vinblastine, leupeptin or a lysosomotropic amine

Kovács, Attila; Reith, Albrecht; Seglen, Per Ottar

doi:10.1016/0014-4827(82)90020-9

Cited by 164 publications

(113 citation statements)

References 32 publications

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“…shown to block the fusion of hepatocellular autophagosomes with lysosomes while only moderately inhibiting the rate of autophagic sequestration, 49,50 thus resulting in an accumulation of autophagosomes to eventually occupy a fraction of about 4% (1/25) of the cytoplasmic volume within two hours. 38 A protein residing exclusively in the autophagosomal delimiting membrane, and not in other cytoplasmic membranes, would thus be enriched about 25x in purified autophagosomes or autophagosomal membranes relative to whole cytoplasm or cytoplasmic membranes.…”

Section: Resultsmentioning

confidence: 99%

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

Øverbye¹,

Fengsrud²,

Seglen³

2007

Autophagy

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140

117

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Section: Resultsmentioning

confidence: 99%

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

Øverbye¹,

Fengsrud²,

Seglen³

2007

Autophagy

Self Cite

140

117

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“…Leupeptin is a general inhibitor of lysosomal protease activity, and although it blocks degradation of AV d (Furuno et al, 1982;Kovacs et al, 1982;Ueno et al, 1991;Yokota et al, 1995), it is not a specific inhibitor of autophagy. To determine whether autophagy is specifically involved in MLB formation, we used 3-MA, which has previously been demonstrated to block autophagy at the initial sequestration step (Seglen and Gordon, 1982).…”

Section: Role Of Autophagy In Mlb Biogenesismentioning

confidence: 99%

Biogenesis of Multilamellar Bodies via Autophagy

Hariri

Millane

Guimond

et al. 2000

MBoC

161

142

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Transfection of Mv1Lu mink lung type II alveolar cells with ␤1-6-N-acetylglucosaminyl transferase V is associated with the expression of large lysosomal vacuoles, which are immunofluorescently labeled for the lysosomal glycoprotein lysosomal-associated membrane protein-2 and the ␤1-6-branched N-glycan-specific lectin phaseolis vulgaris leucoagglutinin. By electron microscopy, the vacuoles present the morphology of multilamellar bodies (MLBs). Treatment of the cells with the lysosomal protease inhibitor leupeptin results in the progressive transformation of the MLBs into electron-dense autophagic vacuoles and eventual disappearance of MLBs after 4 d of treatment. Heterologous structures containing both membrane lamellae and peripheral electron-dense regions appear 15 h after leupeptin addition and are indicative of ongoing lysosome-MLB fusion. Leupeptin washout is associated with the formation after 24 and 48 h of single or multiple foci of lamellae within the autophagic vacuoles, which give rise to MLBs after 72 h. Treatment with 3-methyladenine, an inhibitor of autophagic sequestration, results in the significantly reduced expression of multilamellar bodies and the accumulation of inclusion bodies resembling nascent or immature autophagic vacuoles. Scrape-loaded cytoplasmic FITC-dextran is incorporated into lysosomal-associated membrane protein-2-positive MLBs, and this process is inhibited by 3-methyladenine, demonstrating that active autophagy is involved in MLB formation. Our results indicate that selective resistance to lysosomal degradation within the autophagic vacuole results in the formation of a microenvironment propicious for the formation of membrane lamella. INTRODUCTIONMultilamellar bodies (MLBs) are membrane-bound cellular organelles, which vary in size from 100-2400 nm, are composed of concentric membrane layers, and frequently exhibit an electron-dense core. MLBs are found in numerous cell types where they function in lipid storage and secretion (Schmitz and Mü ller, 1991). In lung type II alveolar cells, MLBs function as secretory granules whose exocytosis results in the deposition of the tubular myelin forms of surfactant on the surface of the alveolae (Hatasa and Nakamura, 1965;Ryan et al., 1975;Williams, 1977). The surfactant film over the alveolar epithelium regulates the surface tension at the air-cell interface and protects the alveola from collapse during respiration (Haagman and van Golde, 1991).Although the secretory function of MLBs in type II alveolar cells is well established, the precise mechanism of MLB biogenesis remains unclear. Autoradiographic studies of murine type II alveolar cells of mouse lungs showed that although phospholipids labeled with [ 3 H]choline are delivered directly from the Golgi to the MLB, proteins metabolically labeled with [ 3 H]leucine are visualized within multivesicular bodies before delivery to MLBs (Chevalier and Collet, 1972). Surfactant proteins A, B, and C are delivered via multivesicular bodies to MLBs, and multivesicular bodies are proposed as th...

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“…The low frequency of autophagy makes it rather difficult to study the process and mechanism of autophagy in normal cells. So far, most of the information about autophagy came from induced autophagy [2][3]. During the past years, we tried to estimate the relative frequency of the autophagy in 32 cell types under normal condition.…”

Section: Introductionmentioning

confidence: 99%

The convergent point of the endocytic and autophagic pathways in leydig cells

Tang

1999

Cell Res

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Endocytic tracers and marker enzyme of lysosomes were used in the present study to analyze the processes of autophagocytosis and endocytosis, and the convergent point of these two pathways in Leydig cells. The endocytic and autophagic compartments can be easily identified in Leydig cells, which makes easier to define the stages of two pathways than was possible before. The evidences indicated that the late endosomes (dense MVBs) deliver their endocytosed gold tracers together with lysosomal enzymes to the early autophagosomes and they are the convergent point of the two pathways. During this convergent process, the early autophagosomes transform into late autophagosomes and the late endosomes transform into mature lysosomes.

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Accumulation of autophagosomes after inhibition of hepatocytic protein degradation by vinblastine, leupeptin or a lysosomotropic amine

Cited by 164 publications

References 32 publications

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

Biogenesis of Multilamellar Bodies via Autophagy

The convergent point of the endocytic and autophagic pathways in leydig cells

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