EEA1 CARRYING VESICLES ARE NOT AUTOPHAGOSOMES IN SERUM-DEPRIVED HeLa CELLS.

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 12­36 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 12­18 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.

[Analysis of vesicle subpopulations carrying early endosomal autoantigen EEA1].

Confocal immunofluorescent analysis of interphase HeLa cells has demonstrated that involved in regulation of homotypic fusions early endosomal autoantigene EEA1 is associated with vesicles represented by two populations differing in apparent size, localization and the level of bound EEA1. Before analysis the cells have been preincubated in serum-deprived medium for 12 h to minimize ligand-dependent endocytosis of serum growth factors. The first subpopulation is mainly represented by large vesicles strongly decorated with EEA1. These vesicles are localized presumably in juxtanuclear region. Microtubule depolimerization experiments have shown that this localization is maintained by tubulin cytoskeleton. The second subpopulation consists of numerous small vesicles slightly stained by EEA1 antibody and localized more peripherally. Double indirect immunofluorescent ananlysis of fixed cell images has revealed that juxtanuclear vesicles enriched in EEA1 are fully colocalized with key protein of early endosomes small GTPase Rab5, whereas about 50% of slightly decorated peripheral vesicles are Rab5-negative. It is found that the number of Rab5-positive vesicles per cell is higher than that of EEA1-positive vesicles. Thus, in serum-deprivated HeLa cells with low endocytic activity two subpopulations of EEA1-vesicles are revealed: the first one carries the both EEA1 at high level and Rab5 (EEA1+++/Rab5+), and the second subpopulation oconsists of weakly decorated EEA1-vesicles, that can be both Rab5-positive and -negative (EEA1+/Rab5- and EEA1+/Rab5+). Besides, there are vesicles carrying Rab5 only (EEA1-/Rab5+). The data obtained favor different functional role of all these subpopulations, which are associated with proteins widely considered as equivalent markers of early endosomes.