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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

Co-segregation of stress-internalized EGFR with markers of pre-melanosomal MVBs.(a) HeLa cells were transfected with 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 with EGFR and either myc (left), fibrillar PMEL (middle) or non-fibrillar PMEL (right). Quantification of co-localization between EGFR (green) and expressed marker (red) for the different conditions is shown below each set of images. Data are mean±s.e.m. of three independent experiments, *P<0.05 and ***P<0.001 (Student's t-test). (b) HeLa cells were treated as above in the presence of 10 nm anti-EGFR-gold (arrows) before preparation for cryo-immunoEM. Ultrathin cryosections were labelled for myc (left), fibrillar PMEL (middle) or non-fibrillar PMEL (right) with 15 nm-gold (arrowheads). Depicted are typical examples of OA1 and fibrillar PMEL+ve MVBs containing stress-internalized but not EGF-stimulated EGFR, and non-fibrillar PMEL containing EGF-stimulated but not stress-internalized EGFR. Quantification of the percentage of EGFR+ve MVBs containing each of the different markers following EGF versus UVC exposure is shown below each set of images. Data are mean±s.e.m. of ≥10 cells, **P<0.01 (Student's t-test). Scale bars, 10 μm for confocal and 100 nm for EM images; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.
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f2: Co-segregation of stress-internalized EGFR with markers of pre-melanosomal MVBs.(a) HeLa cells were transfected with 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 with EGFR and either myc (left), fibrillar PMEL (middle) or non-fibrillar PMEL (right). Quantification of co-localization between EGFR (green) and expressed marker (red) for the different conditions is shown below each set of images. Data are mean±s.e.m. of three independent experiments, *P<0.05 and ***P<0.001 (Student's t-test). (b) HeLa cells were treated as above in the presence of 10 nm anti-EGFR-gold (arrows) before preparation for cryo-immunoEM. Ultrathin cryosections were labelled for myc (left), fibrillar PMEL (middle) or non-fibrillar PMEL (right) with 15 nm-gold (arrowheads). Depicted are typical examples of OA1 and fibrillar PMEL+ve MVBs containing stress-internalized but not EGF-stimulated EGFR, and non-fibrillar PMEL containing EGF-stimulated but not stress-internalized EGFR. Quantification of the percentage of EGFR+ve MVBs containing each of the different markers following EGF versus UVC exposure is shown below each set of images. Data are mean±s.e.m. of ≥10 cells, **P<0.01 (Student's t-test). Scale bars, 10 μm for confocal and 100 nm for EM images; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.

Mentions: We have previously shown that ligand-stimulated EGFR is trafficked in a subset of MVBs11, but the composition and function of non-EGFR-containing MVBs remain poorly characterized. In this respect, melanosomes are lysosome-related organelles generated from a subset of MVBs diverted from the canonical degradative pathway in pigmented cells12. Within those MVBs, premelanosome protein (PMEL) undergoes proteolytic processing to generate fibrils (fibrillar PMEL) upon which melanin is deposited1314, whereas unprocessed non-fibrillar PMEL and cleaved C-terminal PMEL fragments containing its transmembrane domain traffic along the degradative pathway15. We recently showed that the pre-melanosomal G-protein–coupled receptor OA1, when exogenously expressed in HeLa cells, localizes to a subset of MVBs distinct from those carrying ligand-stimulated EGFR16. We now find, by immunofluorescence of HeLa cells transfected with markers of melanosome biogenesis, increased co-localization of EGFR with OA1 and fibrillar PMEL in UVC-exposed, compared with EGF-treated, HeLa cells (Fig. 2a). In contrast, unprocessed non-fibrillar PMEL showed increased co-localization with EGF-bound compared with UVC-exposed EGFR. Parallel cryo-immunoEM showed segregation of stress-exposed EGFR within MVBs containing OA1 and fibrillar PMEL, whereas EGF-bound EGFR segregated within MVBs containing non-fbrillar PMEL and C-terminal PMEL fragments (Fig. 2b and Supplementary Fig. 3a).


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)

Co-segregation of stress-internalized EGFR with markers of pre-melanosomal MVBs.(a) HeLa cells were transfected with 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 with EGFR and either myc (left), fibrillar PMEL (middle) or non-fibrillar PMEL (right). Quantification of co-localization between EGFR (green) and expressed marker (red) for the different conditions is shown below each set of images. Data are mean±s.e.m. of three independent experiments, *P<0.05 and ***P<0.001 (Student's t-test). (b) HeLa cells were treated as above in the presence of 10 nm anti-EGFR-gold (arrows) before preparation for cryo-immunoEM. Ultrathin cryosections were labelled for myc (left), fibrillar PMEL (middle) or non-fibrillar PMEL (right) with 15 nm-gold (arrowheads). Depicted are typical examples of OA1 and fibrillar PMEL+ve MVBs containing stress-internalized but not EGF-stimulated EGFR, and non-fibrillar PMEL containing EGF-stimulated but not stress-internalized EGFR. Quantification of the percentage of EGFR+ve MVBs containing each of the different markers following EGF versus UVC exposure is shown below each set of images. Data are mean±s.e.m. of ≥10 cells, **P<0.01 (Student's t-test). Scale bars, 10 μm for confocal and 100 nm for EM images; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.
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f2: Co-segregation of stress-internalized EGFR with markers of pre-melanosomal MVBs.(a) HeLa cells were transfected with 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 with EGFR and either myc (left), fibrillar PMEL (middle) or non-fibrillar PMEL (right). Quantification of co-localization between EGFR (green) and expressed marker (red) for the different conditions is shown below each set of images. Data are mean±s.e.m. of three independent experiments, *P<0.05 and ***P<0.001 (Student's t-test). (b) HeLa cells were treated as above in the presence of 10 nm anti-EGFR-gold (arrows) before preparation for cryo-immunoEM. Ultrathin cryosections were labelled for myc (left), fibrillar PMEL (middle) or non-fibrillar PMEL (right) with 15 nm-gold (arrowheads). Depicted are typical examples of OA1 and fibrillar PMEL+ve MVBs containing stress-internalized but not EGF-stimulated EGFR, and non-fibrillar PMEL containing EGF-stimulated but not stress-internalized EGFR. Quantification of the percentage of EGFR+ve MVBs containing each of the different markers following EGF versus UVC exposure is shown below each set of images. Data are mean±s.e.m. of ≥10 cells, **P<0.01 (Student's t-test). Scale bars, 10 μm for confocal and 100 nm for EM images; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.
Mentions: We have previously shown that ligand-stimulated EGFR is trafficked in a subset of MVBs11, but the composition and function of non-EGFR-containing MVBs remain poorly characterized. In this respect, melanosomes are lysosome-related organelles generated from a subset of MVBs diverted from the canonical degradative pathway in pigmented cells12. Within those MVBs, premelanosome protein (PMEL) undergoes proteolytic processing to generate fibrils (fibrillar PMEL) upon which melanin is deposited1314, whereas unprocessed non-fibrillar PMEL and cleaved C-terminal PMEL fragments containing its transmembrane domain traffic along the degradative pathway15. We recently showed that the pre-melanosomal G-protein–coupled receptor OA1, when exogenously expressed in HeLa cells, localizes to a subset of MVBs distinct from those carrying ligand-stimulated EGFR16. We now find, by immunofluorescence of HeLa cells transfected with markers of melanosome biogenesis, increased co-localization of EGFR with OA1 and fibrillar PMEL in UVC-exposed, compared with EGF-treated, HeLa cells (Fig. 2a). In contrast, unprocessed non-fibrillar PMEL showed increased co-localization with EGF-bound compared with UVC-exposed EGFR. Parallel cryo-immunoEM showed segregation of stress-exposed EGFR within MVBs containing OA1 and fibrillar PMEL, whereas EGF-bound EGFR segregated within MVBs containing non-fbrillar PMEL and C-terminal PMEL fragments (Fig. 2b and Supplementary Fig. 3a).

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