Abstract
ABSTRACTEndosomal sorting complexes required for transport (ESCRT)-0 sorts ubiquitylated EGFR within the early endosome so that the receptor can be incorporated into intralumenal vesicles. An important question is whether ESCRT-0 acts solely upon EGFR that has already entered the vacuolar early endosome (characterised by the presence of EEA1) or engages EGFR within earlier compartments. Here, we employ a suite of software to determine the localisation of ESCRT-0 at subpixel resolution and to perform particle-based colocalisation analysis with other endocytic markers. We demonstrate that although some of the ESCRT-0 subunit Hrs (also known as HGS) colocalises with the vacuolar early endosome marker EEA1, most localises to a population of peripheral EEA1-negative endosomes that act as intermediates in transporting EGFR from the cell surface to more central early endosomes. The peripheral Hrs-labelled endosomes are distinct from APPL1-containing endosomes, but co-label with the novel endocytic adaptor SNX15. In contrast to ESCRT-0, ESCRT-I is recruited to EGF-containing endosomes at later times as they move to more a central position, whereas ESCRT-III is also recruited more gradually. RNA silencing experiments show that both ESCRT-0 and ESCRT-I are important for the transit of EGF to EEA1 endosomes.
Highlights
The epidermal growth factor receptor (EGFR) pathway serves as an excellent model to study the dynamics of mitogenic receptor signalling and downregulation
Endogenous Hrs localises to peripheral compartments that are depleted of EEA1 The distributions of endogenous Endosomal sorting complexes required for transport (ESCRT)-0 and EEA1 were first examined in HeLaM cells by immunofluorescence microscopy, using a rabbit polyclonal antibody against Hrs and a well-characterised monoclonal antibody against EEA1
This differential localisation of ESCRT-0 and EEA1 was not restricted to HeLaM cells, because strong colocalisation of Hrs and EEA1 was limited to more central endosomes in RPE (Fig. 1A, centre panels) and A549 (Fig. 1A, lower panels) cells
Summary
The epidermal growth factor receptor (EGFR) pathway serves as an excellent model to study the dynamics of mitogenic receptor signalling and downregulation. Received 14 August 2014; Accepted 10 December 2014 which develops into the multivesicular body (MVB) (Raiborg and Stenmark, 2009; Sorkin and Goh, 2009). This morphological alteration is accompanied by changes in the biochemical features and localisation of the endosome, which eventually converts into a late endosome and delivers EGFR directly to the lysosome (Foret et al, 2012; Rink et al, 2005). The precise localisation of these complexes to particular endosomal compartments would contribute to our understanding of the spatial and temporal control of EGFR signalling
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
