ANALYSIS OF VESICLE SUBPOPULATIONS CARRYING EARLY ENDOSOMAL
AUTOANTIGENE EEA1
Ì.V. Zlobina,1 R.S. Êàmentseva,2 Å.S. Kornilova,1,2 Ì.V. Kharchenko 1,*
1 Institute of Cytology RAS, St. Petersburg, and 2 St. Petersburg State University;
* e-mail: mariannakharchenko@gmail.com
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 slighltly 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– è 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.
Key words: early endosomes, ÅÅÀ1, Rab5, confocal immunofluorescence, HeLa cells
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