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Related Experiment Videos

Structural aspects of autophagy

P O Seglen1, T O Berg, H Blankson

  • 1Department of Tissue Culture, Institute for Cancer Research, Norwegian Radium Hospital, Montebello, Oslo, Norway.

Advances in Experimental Medicine and Biology
|January 1, 1996
PubMed
Summary

Researchers purified autophagosomes from rat hepatocytes to study autophagy. They found that intact intermediate filaments are crucial for autophagy, while microtubules and microfilaments play a lesser role.

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Area of Science:

  • Cell Biology
  • Autophagy Research
  • Organelle Biology

Background:

  • Autophagy is a fundamental cellular process for degrading damaged organelles and proteins.
  • Isolation of autophagosomes is crucial for understanding the mechanisms of autophagic sequestration.
  • Previous studies have implicated various cellular components in autophagy, but the precise role of cytoskeletal elements remains debated.

Purpose of the Study:

  • To isolate and characterize autophagosomes from rat hepatocytes as a first step towards understanding phagophore formation.
  • To investigate the role of cytoskeletal elements (intermediate filaments, microtubules, microfilaments) in the process of autophagy.
  • To examine the impact of specific inhibitors and substances on autophagic flux and lysosomal function.

Main Methods:

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  • Purification of autophagosomes from rat hepatocyte homogenates using differential centrifugation and density gradient techniques (Nycodenz, metrizamide).
  • Selective destruction of lysosomes using glycyl-phenylalanyl-naphthylamide (GPN).
  • Cryo-ultrastructural analysis with immunogold labeling to identify autophagic and lysosomal markers.
  • Treatment of hepatocytes with okadaic acid, vinblastine, cytochalasin D, and asparagine to assess their effects on autophagy and lysosomal function.

Main Results:

  • Purified autophagosomes exhibited dual or multiple concentric membranes, suggesting diverse phagophore formation mechanisms.
  • Okadaic acid disrupted the cytokeratin network and completely inhibited autophagy, indicating a dependence on intact intermediate filaments.
  • Vinblastine and cytochalasin D showed only minor inhibition (25-30%) of autophagic sequestration.
  • Vinblastine inhibited autophagic and endocytic flux into lysosomes, reducing lysosomal size.
  • Asparagine treatment led to lysosomal swelling and accumulation of autophagosomes and amphisomes, suggesting inhibition of autophagic flux due to lysosomal dysfunction.

Conclusions:

  • Autophagosome isolation from rat hepatocytes is feasible using established biochemical and biophysical methods.
  • Autophagy appears to be dependent on an intact cytokeratin network (intermediate filaments), with microtubules and microfilaments playing a less significant role.
  • Lysosomal dysfunction, induced by agents like asparagine, can lead to the accumulation of autophagic vacuoles, highlighting the importance of lysosomal function in maintaining autophagic flux.