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Structural Aspects of Autophagy

  • Per O. Seglen
  • Trond Olav Berg
  • Henrietta Blankson
  • Monica Fengsrud
  • Ingunn Holen
  • Per Eivind Strømhaug
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 389)

Summary

As a first step towards isolation of autophagic sequestering membranes (phagophores), we have purified autophagosomes from rat hepatocytes. Lysosomes were selectively destroyed by osmotic rupture, achieved by incubation of hepatocyte homogenates with the cathepsin C substrate glycyl-phenylalanyl-naphthylamide (GPN). Mitochondria and peroxisomes were removed by Nycodenz gradient centrifugation, and cytosol, microsomes and other organelles by rate sedimentation through metrizamide cushions. The purified autophagosomes were bordered by dual or multiple concentric membranes, suggesting that autophagic sequestration might be performed either by single autophagic cisternae or by cisternal stacks.

Okadaic acid, a protein phosphatase inhibitor, disrupted the hepatocytic cytokeratin network and inhibited autophagy completely in intact hepatocytes, perhaps suggesting that autophagy might be dependent on intact intermediate filaments. Vinblastine and cytochalasin D, which specifically disrupted microtubules and microfilaments, respectively, had relatively little (25–30%) inhibitory effect on autophagic sequestration.

In a cryo-ultrastructural study, the various autophagic-lysosomal vacuoles were immunogold-labelled, using the cytosolic enzyme superoxide dismutase as an autophagic marker, Lgpl20 as a lysosomal membrane marker, and bovine serum albumin as an endocytic marker. Vinblastine (50 μM) was found to inhibit both autophagic and endocytic flux into the lysosomes, with a consequent reduction in lysosomal size. Asparagine (20 mM) caused swelling of the lysosomes, probably as a result of the ammonia formation that could be observed at this high asparagine concentration. Autophagosomes and amphisomes (autophagic-endocytic, prelysosomal vacuoles) accumulated in asparagine-treated cells, reflecting an inhibition of autophagic flux that might be a consequence of lysosomal dysfunction.

Keywords

Okadaic Acid Autophagic Vacuole Autophagic Flux Endocytic Marker Nycodenz Gradient 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Plenum Press, New York 1996

Authors and Affiliations

  • Per O. Seglen
    • 1
  • Trond Olav Berg
    • 1
  • Henrietta Blankson
    • 1
  • Monica Fengsrud
    • 1
  • Ingunn Holen
    • 1
  • Per Eivind Strømhaug
    • 1
  1. 1.Department of Tissue Culture, Institute for Cancer ResearchThe Norwegian Radium HospitalOsloNorway

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