Abstract
Early endosomes, regarded as the main sorting station on endocytic pathway, are characterized by high frequency of homotypic fusions mediated by tethering protein EEA1. Despite intensive investigations, biogenesis of endosomes, boundaries between early and late endosomes, and process of cargo transition though them remain obscure. Here, using EGF/EGFR endocytosis as a model and confocal microscopy of fixed and live cells, we provide evidence favoring EEA1-vesicles being pre-existed vesicular compartment, that maintains its resident proteins’ level and is sensitive to biosynthetic, but not endocytic pathway disturbance. EEA1-vesicles directly fuse with incoming EGF/EGFR-vesicles into hybrid endosomes with separated EEA1- and EGFR-domains, thus providing a platform for rapid achievement of an excess of surface-derived membrane that is used to form intraluminal vesicles (ILVs). Thus, multivesicular structures colocalized with EEA1 are still early endosomes. “EEA1-cycle” ends by exclusion of EGFR-containing domains with ILVs inside that turns into MVE and restoration of initial EEA1-vesicles population.
Highlights
Vesicular traffic is one of the basic processes allowing the cell to maintain its homeostasis, respond to external stimuli, coordinate signaling cascades in time and space, etc
As it is seen from MAX projection images these vesicles are tending to concentrate in juxtanuclear area of the cell, and this localization depends on microtubule network as after nocodazole treatment leading to MT depolymerization EEA1-vesicles become scattered throughout the cell, mostly peripherally (Fig 1B)
EEA1-vesicles localization was mostly similar in serum-starved cells and those grown in full medium (Fig 1C)
Summary
Vesicular traffic is one of the basic processes allowing the cell to maintain its homeostasis, respond to external stimuli, coordinate signaling cascades in time and space, etc. Vesicular traffic is a process of multiple macromolecular cargoes transportation from one cell compartment to another by means of membranous transport vesicles [1,2]. Two main highways of protein and lipid flow realizes in all cell types: biosynthetic/exocytic and endocytic ones. Main compartments of the first pathway are Endoplasmic Reticulum (ER) and Golgi Apparatus (GA). Being highly dynamic, these compartments, maintain their identity (i.e. protein/lipid composition, easy recognizable typical morphology, and specific functions), communicate with each other by directed flows of transport vesicles, and are considered as “pre-existing”.
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