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WASH and Tsg101/ALIX-dependent diversion of stress-internalized EGFR from the canonical endocytic pathway.

Tomas A, Vaughan SO, Burgoyne T, Sorkin A, Hartley JA, Hochhauser D, Futter CE - Nat Commun (2015)

Bottom Line: Stress-internalized EGFR is retained intracellularly by continued p38 activity in a mechanism involving ubiquitin-independent, ESCRT/ALIX-dependent incorporation onto intraluminal vesicles (ILVs) of MVBs.In contrast to the internalization-independent EGF-stimulated activation, UVC/cisplatin-triggered EGFR activation depends on EGFR internalization and intracellular retention.EGFR signalling from this MVB subpopulation delays apoptosis and might contribute to chemoresistance.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Cell Biology, UCL Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK [2].

ABSTRACT
Stress exposure triggers ligand-independent EGF receptor (EGFR) endocytosis, but its post-endocytic fate and role in regulating signalling are unclear. We show that the p38 MAP kinase-dependent, EGFR tyrosine kinase (TK)-independent EGFR internalization induced by ultraviolet light C (UVC) or the cancer therapeutic cisplatin, is followed by diversion from the canonical endocytic pathway. Instead of lysosomal degradation or plasma membrane recycling, EGFR accumulates in a subset of LBPA-rich perinuclear multivesicular bodies (MVBs) distinct from those carrying EGF-stimulated EGFR. Stress-internalized EGFR co-segregates with exogenously expressed pre-melanosomal markers OA1 and fibrillar PMEL, following early endosomal sorting by the actin polymerization-promoting WASH complex. Stress-internalized EGFR is retained intracellularly by continued p38 activity in a mechanism involving ubiquitin-independent, ESCRT/ALIX-dependent incorporation onto intraluminal vesicles (ILVs) of MVBs. In contrast to the internalization-independent EGF-stimulated activation, UVC/cisplatin-triggered EGFR activation depends on EGFR internalization and intracellular retention. EGFR signalling from this MVB subpopulation delays apoptosis and might contribute to chemoresistance.

No MeSH data available.


Related in: MedlinePlus

Sorting of EGF-bound and stress-internalized EGFR onto separate MVB subsets is WASH dependent.(a) Control flox/flox and WASH knock-out (WASHOUT) MEFs transfected with human EGFR were exposed to UVC and incubated for 1 h to allow stress-induced EGFR internalization in the presence of anti-EGFR 108 antibody. Cells were washed, incubated with EGF-AlexaFluor 488 for 30 min (red), and processed for immunofluorescence with an AlexaFluor 555 secondary antibody to label EGFR (green). Both EGF+ve and EGF-ve EGFR-containing endosomes are present in control flox/flox, but these are largely merged in WASHOUT MEFs. (b) Control flox/flox and WASHOUT MEFs were co-transfected with human EGFR and OA1-myc or PMEL, and either treated with EGF for 30 min or exposed to UVC and incubated for 1 h. Cells were co-stained for EGFR (green) and the following markers (in red): myc (top panels), fibrillar PMEL (central panels) or non-fibrillar PMEL (bottom panels). (c) Quantification of co-localization between EGFR and expressed markers for the different conditions. Data are mean±s.e.m. of three independent experiments, ***P<0.001 (Student's t-test). Scale bars, 10 μm; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.
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f3: Sorting of EGF-bound and stress-internalized EGFR onto separate MVB subsets is WASH dependent.(a) Control flox/flox and WASH knock-out (WASHOUT) MEFs transfected with human EGFR were exposed to UVC and incubated for 1 h to allow stress-induced EGFR internalization in the presence of anti-EGFR 108 antibody. Cells were washed, incubated with EGF-AlexaFluor 488 for 30 min (red), and processed for immunofluorescence with an AlexaFluor 555 secondary antibody to label EGFR (green). Both EGF+ve and EGF-ve EGFR-containing endosomes are present in control flox/flox, but these are largely merged in WASHOUT MEFs. (b) Control flox/flox and WASHOUT MEFs were co-transfected with human EGFR and OA1-myc or PMEL, and either treated with EGF for 30 min or exposed to UVC and incubated for 1 h. Cells were co-stained for EGFR (green) and the following markers (in red): myc (top panels), fibrillar PMEL (central panels) or non-fibrillar PMEL (bottom panels). (c) Quantification of co-localization between EGFR and expressed markers for the different conditions. Data are mean±s.e.m. of three independent experiments, ***P<0.001 (Student's t-test). Scale bars, 10 μm; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.

Mentions: The finding that OA1 and fibrillar PMEL co-segregate with stress-stimulated EGFR provides a means of monitoring the diversion of stress- and ligand-stimulated EGFR from early endosomes. Branched early endosomal actin networks resulting from Arp2/3 activation by the endosomal actin polymerization-promoting complex, WASH17, have recently been found to play a vital role in sorting of specific endosomal cargoes to different destinations18, such as retromer-mediated endosome-to-Golgi retrieval19 and plasma membrane recycling20. The WASH complex has also been shown to associate with BLOC-1 (biogenesis of lysosomal organelles complex-1), required for selective cargo exit from early endosomes to melanosomes21, suggesting that WASH-mediated actin polymerization may function in melanosome biogenesis22. We found that WASH is required for segregation of stress-internalized EGFR away from EGF-bound EGFR by comparing mouse embryonic fibroblasts (MEFs) derived from control flox/flox and Cre-mediated conditional WASH knockout (WASHOUT) embryos23. In flox/flox MEFs treated first with UVC and then fluorescent EGF, there are two populations (EGF positive and EGF negative) of EGFR present (Fig. 3a). However, in WASHOUT MEFs, EGFR and EGF signals are totally merged in the same endosomal compartments. Furthermore, although in flox/flox MEFs (like in HeLa cells) UVC-exposed EGFR shows greater co-localization with OA1 and fibrillar PMEL than EGF-bound EGFR, in WASHOUT MEFs UVC-exposed and EGF-bound EGFR co-stain equally well with these markers (Fig. 3b, quantified in Fig. 3c). Similarly, in flox/flox MEFs, EGF-bound EGFR shows greater co-localization with non-fibrillar PMEL than UVC-exposed receptor, but this difference is lost in WASHOUT MEFs. The requirement for the WASH complex in segregation of stress-internalized EGFR along with pre-melanosomal factors away from ligand-stimulated EGFR contrasts with the unimpaired lysosomal degradation of EGF-bound EGFR and plasma membrane recycling of TfR in WASH knockouts23.


WASH and Tsg101/ALIX-dependent diversion of stress-internalized EGFR from the canonical endocytic pathway.

Tomas A, Vaughan SO, Burgoyne T, Sorkin A, Hartley JA, Hochhauser D, Futter CE - Nat Commun (2015)

Sorting of EGF-bound and stress-internalized EGFR onto separate MVB subsets is WASH dependent.(a) Control flox/flox and WASH knock-out (WASHOUT) MEFs transfected with human EGFR were exposed to UVC and incubated for 1 h to allow stress-induced EGFR internalization in the presence of anti-EGFR 108 antibody. Cells were washed, incubated with EGF-AlexaFluor 488 for 30 min (red), and processed for immunofluorescence with an AlexaFluor 555 secondary antibody to label EGFR (green). Both EGF+ve and EGF-ve EGFR-containing endosomes are present in control flox/flox, but these are largely merged in WASHOUT MEFs. (b) Control flox/flox and WASHOUT MEFs were co-transfected with human EGFR and OA1-myc or PMEL, and either treated with EGF for 30 min or exposed to UVC and incubated for 1 h. Cells were co-stained for EGFR (green) and the following markers (in red): myc (top panels), fibrillar PMEL (central panels) or non-fibrillar PMEL (bottom panels). (c) Quantification of co-localization between EGFR and expressed markers for the different conditions. Data are mean±s.e.m. of three independent experiments, ***P<0.001 (Student's t-test). Scale bars, 10 μm; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.
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f3: Sorting of EGF-bound and stress-internalized EGFR onto separate MVB subsets is WASH dependent.(a) Control flox/flox and WASH knock-out (WASHOUT) MEFs transfected with human EGFR were exposed to UVC and incubated for 1 h to allow stress-induced EGFR internalization in the presence of anti-EGFR 108 antibody. Cells were washed, incubated with EGF-AlexaFluor 488 for 30 min (red), and processed for immunofluorescence with an AlexaFluor 555 secondary antibody to label EGFR (green). Both EGF+ve and EGF-ve EGFR-containing endosomes are present in control flox/flox, but these are largely merged in WASHOUT MEFs. (b) Control flox/flox and WASHOUT MEFs were co-transfected with human EGFR and OA1-myc or PMEL, and either treated with EGF for 30 min or exposed to UVC and incubated for 1 h. Cells were co-stained for EGFR (green) and the following markers (in red): myc (top panels), fibrillar PMEL (central panels) or non-fibrillar PMEL (bottom panels). (c) Quantification of co-localization between EGFR and expressed markers for the different conditions. Data are mean±s.e.m. of three independent experiments, ***P<0.001 (Student's t-test). Scale bars, 10 μm; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.
Mentions: The finding that OA1 and fibrillar PMEL co-segregate with stress-stimulated EGFR provides a means of monitoring the diversion of stress- and ligand-stimulated EGFR from early endosomes. Branched early endosomal actin networks resulting from Arp2/3 activation by the endosomal actin polymerization-promoting complex, WASH17, have recently been found to play a vital role in sorting of specific endosomal cargoes to different destinations18, such as retromer-mediated endosome-to-Golgi retrieval19 and plasma membrane recycling20. The WASH complex has also been shown to associate with BLOC-1 (biogenesis of lysosomal organelles complex-1), required for selective cargo exit from early endosomes to melanosomes21, suggesting that WASH-mediated actin polymerization may function in melanosome biogenesis22. We found that WASH is required for segregation of stress-internalized EGFR away from EGF-bound EGFR by comparing mouse embryonic fibroblasts (MEFs) derived from control flox/flox and Cre-mediated conditional WASH knockout (WASHOUT) embryos23. In flox/flox MEFs treated first with UVC and then fluorescent EGF, there are two populations (EGF positive and EGF negative) of EGFR present (Fig. 3a). However, in WASHOUT MEFs, EGFR and EGF signals are totally merged in the same endosomal compartments. Furthermore, although in flox/flox MEFs (like in HeLa cells) UVC-exposed EGFR shows greater co-localization with OA1 and fibrillar PMEL than EGF-bound EGFR, in WASHOUT MEFs UVC-exposed and EGF-bound EGFR co-stain equally well with these markers (Fig. 3b, quantified in Fig. 3c). Similarly, in flox/flox MEFs, EGF-bound EGFR shows greater co-localization with non-fibrillar PMEL than UVC-exposed receptor, but this difference is lost in WASHOUT MEFs. The requirement for the WASH complex in segregation of stress-internalized EGFR along with pre-melanosomal factors away from ligand-stimulated EGFR contrasts with the unimpaired lysosomal degradation of EGF-bound EGFR and plasma membrane recycling of TfR in WASH knockouts23.

Bottom Line: Stress-internalized EGFR is retained intracellularly by continued p38 activity in a mechanism involving ubiquitin-independent, ESCRT/ALIX-dependent incorporation onto intraluminal vesicles (ILVs) of MVBs.In contrast to the internalization-independent EGF-stimulated activation, UVC/cisplatin-triggered EGFR activation depends on EGFR internalization and intracellular retention.EGFR signalling from this MVB subpopulation delays apoptosis and might contribute to chemoresistance.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Cell Biology, UCL Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK [2].

ABSTRACT
Stress exposure triggers ligand-independent EGF receptor (EGFR) endocytosis, but its post-endocytic fate and role in regulating signalling are unclear. We show that the p38 MAP kinase-dependent, EGFR tyrosine kinase (TK)-independent EGFR internalization induced by ultraviolet light C (UVC) or the cancer therapeutic cisplatin, is followed by diversion from the canonical endocytic pathway. Instead of lysosomal degradation or plasma membrane recycling, EGFR accumulates in a subset of LBPA-rich perinuclear multivesicular bodies (MVBs) distinct from those carrying EGF-stimulated EGFR. Stress-internalized EGFR co-segregates with exogenously expressed pre-melanosomal markers OA1 and fibrillar PMEL, following early endosomal sorting by the actin polymerization-promoting WASH complex. Stress-internalized EGFR is retained intracellularly by continued p38 activity in a mechanism involving ubiquitin-independent, ESCRT/ALIX-dependent incorporation onto intraluminal vesicles (ILVs) of MVBs. In contrast to the internalization-independent EGF-stimulated activation, UVC/cisplatin-triggered EGFR activation depends on EGFR internalization and intracellular retention. EGFR signalling from this MVB subpopulation delays apoptosis and might contribute to chemoresistance.

No MeSH data available.


Related in: MedlinePlus