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

Abstract

According to current model, stimulation of EGF-receptor endocytosis results in recruitment onto early endosomes cytosolic tether protein EEA1 necessary for their further fusions. However, EEA1-positive vesicles are found in the cells not treated with growth factor, that were incubated in serum-free conditions. It is known also that prolonged serum deprivation induces autophagosomes formation, the process possibly involving endocytic compartments. To check whether EEA1-positive vesicles seen in serum-deprived HeLa cells are autophagosomes, we here evaluated colocalization of EEA1 and autophagosome marker LC3 and studied dynamics of the EEA1- and LC3-vesicles’ number and size during 12—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 show that serum starvation results in increase of only mean autophagosomes’ size, while the number and size of EEA1-vesicles did not changed. Colocalization of EEA1 and LC3 in serum-free cells was very low during first 12—18 h of starvation and increased insignificantly only by 36 h. Biosynthetic pathway inhibition by Golgi apparatus disruption by brefeldin A, decreased the number and increased the size of EEA1-vesicles. LC3-vesicles also demonstrated an increase of mean size and growth of colocalization with EEA1. Thus, we conclude that the majority of EEA1-vesicles in serum-starved cells are not autophagosomes. More pronounced effect of brefeldin A indicates that blockade of biosynthetic pathway is more strong stress factor comparing to serum deprivation in HeLa cells. This also suggests that this pathway is involved in EEA1-vesicles biogenesis.