EEA1-carrying vesicles are not autophagosomes in serum-deprived HeLa cells

Аннотация

According to the current model, stimulation of EGF-receptor endocytosis results in recruitment of the cytosolic tether protein EEA1 onto early endosomes necessary for their further fusion. However, EEA1-positive vesicles are found in cells not treated with growth factor and incubated under serum-free conditions. Prolonged serum deprivation, which is known to induce the formation of autophagosomes, may involve endocytic compartments. To check whether EEA1-positive vesicles seen in serum-deprived HeLa cells are autophagosomes, we have evaluated colocalization of EEA1 and autophagosome marker LC3, as well as changes in the number and size of EEA1 and LC3 vesicles in the course of 12- to 36-h cell cultivation in serum-free medium. It was found that the number of autophagosomes per cell is significantly less than the number of EEA1 vesicles. We showed that serum starvation resulted in an increase of average size of autophagosomes, while the number and size of EEA1 vesicles remained unchanged. Colocalization of EEA1 and LC3 in serum-free medium was very low during the first 12–18 h of starvation and increased only slightly by 36 h. Biosynthetic pathway inhibition by the disruption of Golgi apparatus with Brefeldin A resulted in a decrease in the number and increased the size of EEA1 vesicles. LC3 vesicles also exhibited an increase of average size and colocalization with EEA1. Thus, we conclude that the majority of EEA1 vesicles in serum-starved cells are not autophagosomes. The prominent effect of Brefeldin A indicates that blockage of the biosynthetic pathway is a stronger stress factor than is serum deprivation for HeLa cells. This also suggests that this pathway is involved in EEA1 vesicle biogenesis.