Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes.
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.
Köchl
Robert
R
Cancer Research UK, London Research Institute, Secretory Pathways Laboratory, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK.
Hu
Xiao Wen
XW
Chan
Edmond Y W
EY
Tooze
Sharon A
SA
D000818:Animals; D001343:Autophagy / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003470:Culture Media; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology; D008854:Microscopy, Electron; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D015739:Nocodazole / Q000494:pharmacology; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014747:Vinblastine / Q000494:pharmacology
D003470:Culture Media; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D014747:Vinblastine; D015739:Nocodazole