title: ESCRT-0 marks an APPL1-independent transit route for EGFR between the cell surface and the EEA1-positive early endosome.
abstract: Endosomal 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.
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK Children's Medical Research Institute, 214 Hawkesbury Road, Westmead, NSW 2145, Australia.
authors.forename: Neftali
authors.initials: N
authors.lastname: Flores-Rodriguez
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.
authors.forename: David A
authors.initials: DA
authors.lastname: Kenwright
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.
authors.forename: Pei-Hua
authors.initials: PH
authors.lastname: Chung
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Harrison
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.
authors.forename: Flavia
authors.initials: F
authors.lastname: Stefani
authors.affiliation: School of Physics and Astronomy, University of Manchester, Manchester M13 9PT, UK Photon Science Institute, University of Manchester, Manchester M13 9PT, UK.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Waigh
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK Photon Science Institute, University of Manchester, Manchester M13 9PT, UK viki.allan@manchester.ac.uk philip.woodman@manchester.ac.uk.
authors.forename: Victoria J
authors.initials: VJ
authors.lastname: Allan
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK viki.allan@manchester.ac.uk philip.woodman@manchester.ac.uk.
authors.forename: Philip G
authors.initials: PG
authors.lastname: Woodman
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000502:physiology*; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering; D058305:Sorting Nexins / Q000378:metabolism; D022161:Transport Vesicles / Q000378:metabolism; D054875:Ubiquitination; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C464801:APPL1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D056827:Endosomal Sorting Complexes Required for Transport; D010750:Phosphoproteins; D034741:RNA, Small Interfering; C429023:SNX15 protein, human; C109775:STAM protein, human; C491647:STAM2 protein, human; D058305:Sorting Nexins; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; D004815:Epidermal Growth Factor; C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: EEA1; EGFR; ESCRT-I; Hrs; Particle-based colocalisation