Autophagosome maturation in primary rat hepatocytes
Nutrient deprivation of eukaryotic cells elicits a rapid survival response, including the induction of autophagy. Autophagy, or "self-eating", involves the formation of autophagosomes from an unknown membrane source and the sequestration of cytosolic components, including organelles such as mitochondria, endoplasmic reticulum and small vesicles. Autophagosomes then fuse with protease-containing endosomes and their contents are degraded. This allows the cell to recycle amino acids and to reuse them for the synthesis of new proteins. To study the formation and fusion of autophagosomes, I have developed in vivo and in vitro assays, based on primary rat hepatocytes cultures. For the in vitro assay, the aim of which is to identify proteins involved in fusion, I have designed specific markers and internalized them into endosomes and autophagosomes. Both vesicle populations have been purified and used to reconstitute fusion in vitro. For the in vivo experiments I expressed the GFP-tagged autophagosomal marker, LC3, in cultured primary rat hepatocytes. By measuring the translocation of GFP-LC3 from a cytosolic pool to newly formed autophagosomes, using a high throughput-imaging system, and by assaying for the lipidation of GFP-LC3, I was able to quantify the rate and magnitude of autophagosome formation and fusion. Starvation led to an increase in the rate of autophagosome formation, and the total number of autophagosomes per cell increased more than two-fold. Autophagosome formation was independent of mTOR, a negative regulator of autophagy in yeast and many cell lines, and could be strongly inhibited by leucine, a regulatory amino acid. I then investigated the role of microtubules in the formation and fusion of autophagosomes, using the microtubule-depolymerising drugs nocodazole and vinblastine. I found that nocodazole treatment reduced the rate of autophagosome formation and completely inhibited their mobility. In addition, both drugs inhibited fusion with endosomes, showing that an intact microtubule network is also required for fusion. Interestingly, vinblastine also strongly stimulated autophagosome formation, even in nutrient-rich medium. This effect was independent of mTOR activity, but required the autophagy proteins Atg5 and Atg6, suggesting that vinblastine affects a novel signalling pathway upstream of autophagy proteins.