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First published online July 12, 2005
doi: 10.1242/10.1242/jcs.02421

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Depletion of TSG101 forms a mammalian `Class E' compartment: a multicisternal early endosome with multiple sorting defects

Aurelie Doyotte^1,*, Matthew R. G. Russell^2,*, Colin R. Hopkins² and Philip G. Woodman^1,

¹ Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK
² Department of Biological Sciences, Imperial College, London, SW7 2AS, UK

View larger version (16K): [in a new window]   Fig. 1. Depletion of TSG101 causes intracellular retention of undegraded EGF. (A) Duplicate samples of lamin A-depleted (L) or TSG101-depleted (T) cells were lysed in SDS-PAGE sample buffer and analysed by western blotting for TSG101, VPS28, and tubulin. (B) Lamin A-depleted (upper panel) or TSG101-depleted (lower panel) HeLa cells were incubated with 125I-EGF for 15 minutes at 37°C. After a brief wash to remove cell surface 125I-EGF, cells were incubated at 37°C for the indicated times. Samples were analysed for degraded 125I-EGF (circles), undegraded recycled EGF (triangles) and remaining cell-associated 125I-EGF (squares). Note that initial binding of EGF was reduced by 27.4%±10.0% in TSG101-depleted cells. Values are means from three experiments, each performed in duplicate, ± s.e.m.

View larger version (50K): [in a new window]   Fig. 2. EGF accumulates in aberrant early endosomes in TSG101-depleted cells. (A-C) Lamin A-depleted HeLa cells were allowed to internalise Alexa Fluor488-labelled EGF for 3 hours and cells were examined by fluorescence microscopy for EEA1 (A), Hrs (B) or LAMP-2 (C) (note that virtually all the Alexa Fluor488-labelled EGF had been lost from cells at this time, and panels showing the residual Alexa Fluor488-labelled EGF are not provided). (D-L) TSG101-depleted cells were allowed to internalise Alexa Fluor488-labelled EGF for 3 hours. Cells were examined by fluorescence microscopy for EGF (D,G,J; green), EEA1 (E; red), Hrs (H; red) or LAMP-2 (K; red). Merged images are shown in F,I,L. Bar, 10 µm.

View larger version (68K): [in a new window]   Fig. 3. Clathrin and dynamin do not associate with aberrant early endosomes induced by TSG101 depletion. TSG101-depleted HeLa cells were allowed to internalise Alexa Fluor488-labelled EGF for 3 hours at 37°C. Cells were fixed and examined by fluorescence microscopy for EGF (A,D; green), clathrin (B; red) or dynamin (E; red). Merged images are shown in C and F. Bar, 10 µm.

View larger version (146K): [in a new window]   Fig. 4. Morphology of multicisternal early endosomes in TSG101-depleted cells. TSG101-depleted HeLa cells were incubated with TfHRP for 30 minutes to label early endosomes (A,C), or incubated with EGF and 108 anti-EGFR 10 nm gold for 30 minutes at 4°C, washed, and incubated at 37°C for a further 3 hours, with TfHRP added for the final 30 min (B; note that in this oblique section the inter-cisternal matrix appears as striations). Arrowheads indicate anti-EGFR gold. Arrows indicate matrix. Lamin A-depleted HeLa cells were incubated with EGF and 108 anti-EGFR 10 nm gold for 30 minutes at 4°C, washed, and incubated at 37°C for a further 30 minutes (D) or 3 hours (E). For the final 30 min the cells were incubated with Tf-HRP. Electron dense content; cross linked Tf-HRP. Bars, 0.1 µm (A,D,E); 0.5 µm (B,C).

View larger version (92K): [in a new window]   Fig. 5. Hrs and SNX1 localise to multicisternal endosomes. TSG101-depleted (A,B) and lamin A-depleted (C) HeLa cells were incubated with EGF and 108 anti-EGFR 10 nm gold for 30 minutes at 4°C, washed, and incubated at 37°C for a further 1 hour (for the experiment showing Hrs labelling) or 3 hours (for the experiment showing SNX1), with TfHRP added for the final 30 minutes. The cells were then post-fix labelled with 5 nm gold for Hrs (A) or SNX1 (B, C). Arrowheads indicate anti-EGFR-gold; arrows indicate anti-Hrs-gold or anti-SNX1-gold. Bars, 0.5 µm (A); 0.1 µm (B,C).

View larger version (143K): [in a new window]   Fig. 6. Lgp120 does not localise to multicisternal endosomes. TSG101-depleted (A) and lamin A-depleted (B) HeLa cells were incubated with EGF and 108 anti-EGFR 10 nm gold for 30 minutes at 4°C, washed, and incubated at 37°C for a further 1 hour, with TfHRP added for the final 30 minutes. The cells were then post-fix labelled with 5 nm gold for lgp120. Arrowheads indicate EGFR-108 gold and arrows indicate matrix. The boxed region in A is shown at a higher magnification in the inset, which has been altered to enhance contrast. Bars, 0.5 µm (A,B) 0.1 µm (inset).

View larger version (160K): [in a new window]   Fig. 7. Transport to lysosomes is impaired in TSG101-depleted cells. (A) TSG101-depleted HeLa cells were incubated with EGF and 108 anti-EGFR 10 nm gold for 30 minutes at 4°C, washed, and incubated at 37°C for a further 3 hours, with TfHRP added for the final 30 minutes. Examples of EGFR-gold complexes are indicated by arrows. (B,C) Lamin A-depleted control (B) or TSG101-depleted (C) HeLa cells were incubated for 8 hours with fluid-phase HRP at 37°C. Similar results were obtained after 4 and 6 hours (not shown). Electron dense content is crosslinked HRP. Lysosomes (based on morphology) are indicated by arrowheads. Bars, 0.5 µm.

View larger version (158K): [in a new window]   Fig. 8. EGFR and TR colocalise to aberrant early endosomes. TSG101-depleted (A) and lamin A-depleted (B,C) HeLa cells were incubated with EGF and 108 anti-EGFR 10 nm gold for 30 minutes at 4°C, washed, and incubated at 37°C for a further 1 hour, with TfHRP added for the final 30 minutes. The cells were then post-fix labelled with 5 nm gold for TR. Note that B and C are from the same cell labelled for EGFR and TR and show the separate localisations of EGFR (B) and TR (C). EGFR-108 gold complexes are indicated by arrows (10 nm gold). Bars, 0.1 µm.

View larger version (66K): [in a new window]   Fig. 9. Depletion of TSG101 disrupts trafficking of lysosomal precursors at the early endosome. (A) Lamin A or TSG101-depleted HeLa cells were pulse labelled with [35S]methionine and chased as indicated in the presence of excess unlabelled methionine. Anti-cathepsin D immunoprecipitates from cell culture supernatants (top part) and cells (bottom part) were analysed by SDS-PAGE and fluorography. The migration positions of procathepsin D (P; 45 kDa), the intermediate cleaved form of cathepsin D (I; 40 kDa) and mature cathepsin D (M; 27 kDa) are indicated. Duplicate samples from a representative experiment are shown. (B) Phosphorimager data from cathepsin D pulse-chase experiments were analysed to determine the amounts of cathepsin D cleaved to the intermediate form (interm), cleaved to the mature form (mature) or secreted from cells (secreted) in TSG101-depleted cells relative to lamin A-depleted cells. Data from a 2-hour chase are shown as white bars, and those from a 4-hour chase as shaded bars. Data are the means ± s.e.m. from three independent experiments, each performed in duplicate. (C) Lamin A-depleted (upper panels) or TSG101-depleted (centre panels) cells were fixed and stained for mannose 6-phosphate receptor (M6PR; green), and GM130 or EEA1 (red) as indicated. Inserts are magnified twofold relative to the main images. Alternatively, TSG101-depleted cells (lower panels) were allowed to internalise Alexa Fluor488-labelled EGF for 3 hours at 37°C prior to fixation, then examined for EGF (green) and mannose 6-phosphate receptor (red). Bar, 10 µm.

View larger version (110K): [in a new window]   Fig. 10. Recycling pathways to the TGN and plasma membrane are disrupted by TSG101 depletion. (A) Lamin A-depleted (panels A-C,G-I) or TSG101-depleted (panels D-F,J-L) HeLa cells were fixed directly (A-F) and stained for Golgin-84 (green; A,D) and TGN46 (red; B,E), or allowed to internalise Alexa Fluor488-labelled EGF for 3 hours at 37°C prior to fixation (G-L), then examined by fluorescence microscopy for EGF (green; G,J) and transferrin receptor (red; H,K). Merged images are shown in panels C,F,I,L. Bar, 10 µm. (B) Upper panel: 125I-Tf was bound to lamin A-depleted or TSG101-depleted HeLa cells for 30 minutes at 4°C, after which the cells were washed to remove unbound material and warmed to 37°C in the presence of excess unlabelled Tf for the indicated times. Cells were chilled on ice and washed with a deferoxamine cycle to remove residual surface-bound Tf before being extracted in 1 M NaOH and counted for radioactivity. Results are expressed as the percentage of Tf that remained cell associated relative to that which had been initially bound. Results are the means from two experiments each performed in duplicate ± s.d. Lower panel: results (expressed as cpm ± s.d.) from samples to which 125I-Tf was bound at 4°C but not subjected to a deferoxamine wash. (C) Lamin A-depleted (L) or TSG101-depleted (T) HeLa cells were western blotted for TSG101 and TR. (D) Control (upper panel) and TSG101-depleted (lower panel) cells were incubated with Texas red-conjugated Tf for 60 minutes at 4°C followed by 5 minutes at 37°C, then fixed and examined by confocal microscopy. The photomultiplier and image analysis settings were identical in both samples. Bar, 10 µm.

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