Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes

Robert Köchl, Xiao Wen Hu, Edmond Y W Chan, Sharon A Tooze

Research output: Contribution to journalArticle

282 Citations (Scopus)

Abstract

Nutrient deprivation of eukaryotic cells provokes a variety of stress responses, including autophagy. Autophagy is carried out by autophagosomes which sequester cytosolic components and organelles for degradation after fusion with protease-containing endosomes. To determine the role of microtubules in autophagy, we used nocodazole and vinblastine to disrupt microtubules and independently measured formation and fusion of autophagsosomes in primary rat hepatocytes. By measuring the translocation of GFP-LC3, an autophagosomal marker, to autophagosomes and the lipidation of GFP-LC3, we quantified the rate and magnitude of autophagosome formation. Starvation increased both the rate of autophagosome formation over the basal level and the total number of autophagosomes per cell. Maximal autophagosome formation required an intact microtubule network. Fusion of autophagosomes with endosomes, assayed by acquisition of protease-inhibitor sensitivity as well as overlap with LysoTracker Red-positive endosomes, required intact microtubules. Live-cell imaging demonstrated that autophagosomes were motile structures, and their movement also required microtubules. Interestingly, vinblastine stimulated autophagosome formation more than twofold before any discernable change in the microtubule network was observed. Stimulation of autophagosome formation by vinblastine was independent of nutrients and mTOR activity but was inhibited by depletion of the Autophagy proteins Atg5 and Atg6, known to be required for autophagy.

LanguageEnglish
Pages129-145
Number of pages17
JournalTraffic
Volume7
Issue number2
Early online date5 Dec 2005
DOIs
Publication statusPublished - Feb 2006

Fingerprint

Vinblastine
Endosomes
Microtubules
Fusion reactions
Nutrients
Autophagy
Nocodazole
Protease Inhibitors
Rats
Peptide Hydrolases
Imaging techniques
Degradation
Autophagosomes
Proteins
Food
Eukaryotic Cells
Starvation
Organelles
Hepatocytes

Keywords

  • animals
  • autophagy
  • cells, cultured
  • culture media
  • endosomes
  • green fluorescent proteins
  • hepatocytes
  • membrane fusion
  • microscopy, electron
  • microtubules
  • nocodazole
  • phagosomes
  • rats
  • recombinant fusion proteins
  • vinblastine

Cite this

Köchl, Robert ; Hu, Xiao Wen ; Chan, Edmond Y W ; Tooze, Sharon A. / Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes. In: Traffic. 2006 ; Vol. 7, No. 2. pp. 129-145.
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Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes. / Köchl, Robert; Hu, Xiao Wen; Chan, Edmond Y W; Tooze, Sharon A.

In: Traffic, Vol. 7, No. 2, 02.2006, p. 129-145.

Research output: Contribution to journalArticle

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