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First published online 24 May 2005
doi: 10.1242/jcs.02382

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Alix regulates cortical actin and the spatial distribution of endosomes

Department of Biochemistry, the Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway

View larger version (35K): [in a new window]   Fig. 1. Downregulation of Alix expression in HeLa cells by siRNA. (A) HeLa cells were treated with control RNA (–) or with siRNA against Alix (+) for the incubation times indicated. The cells were lysed and analysed by SDS/PAGE and inmunoblotting with anti-Alix antibodies. Anti--tubulin antibodies were used to verify equal loadings. (B) Same experiment as in A, but the incubation time was 72 hours in the presence of siRNA, followed by 72 hours without siRNA. The amounts of Alix, Hrs, Tsg101 and -tubulin in cell lysates are shown.

View larger version (109K): [in a new window]   Fig. 2. Alix depletion alters the distribution of endosomes. HeLa cells were treated with control RNA (A-D,J) or with siRNA against Alix (E-I) as in Fig. 1B. The cells were permeabilised before fixation and were stained with anti-EEA1 (A,E,I,J), anti-Hrs (B,F), anti-LBPA (C,G), or anti-LAMP1 (D,H). Scale bars: 5 µm. Asterisks in I,J represent nuclei.

View larger version (60K): [in a new window]   Fig. 3. Alix depletion increases de colocalisation of early and late endosomal markers in the perinuclear region. HepII cells were treated with control RNA (A-D) or with siRNA against Alix (E-H) as in Fig. 1B. The cells were permeabilised before fixation and were stained with anti-EEA1 (A,E), anti-Hrs (B,F) and anti-CD63 (C,G). Scale bars: 5 µm.

View larger version (76K): [in a new window]   Fig. 4. Alix depletion alters the distribution of transferrin-positive recycling endosomes. HeLa cells were treated with control RNA (A-D) or with siRNA against Alix (E-H) as in Fig. 1B. The cells were incubated with Tf-Alexa594 at 37°C for 5 minutes followed by a 15-minute chase to label the recycling endosomes (A,E). The cells were fixed before permeabilisation and were stained with anti-EEA1 (B,F) and anti-Rab11 (C,G). D and H are merged images of B,C and F,G, respectively. White colour indicates colocalisation between Tf-Alexa594, EEA1 and Rab11. Scale bars: 5 µm.

View larger version (17K): [in a new window]   Fig. 5. Effect of Alix depletion on transferrin endocytosis and recycling. HeLa cells were treated with control RNA or with siRNA against Alix as in Fig. 1B. For the endocytosis experiments (A), cells were incubated with Ru-tag-Tf for different times at 37°C as described in the Materials and Methods, and the surface-bound Ru-tag-Tf was reduced with MESNA. The amount of cell-associated Tf was measured using an ORIGEN analyser, and endocytosed Tf is presented as a percentage of total cell-associated Tf. Error bars represent the s.e.m. of three independent experiments performed in duplicate. (B) Cells were incubated with Ru-tag-Tf for 10 minutes and then washed and chased at 37°C for 5, 10 or 15 minutes before MESNA treatment. Error bars represent the s.e.m. of three independent experiments performed in duplicate.

View larger version (24K): [in a new window]   Fig. 6. Depletion of Alix does not inhibit EGF receptor downregulation. HeLa cells treated with control RNA (–) or with siRNA (+) against Alix (see Fig. 1B) or with siRNA (+) against HCRP1. The cells were stimulated for 30 minutes with EGF 200 ng/ml (time 0), and then washed and chased for 6 hours in the presence of 10 µg/ml cycloheximide before the cells were lysed in lysis buffer. The lysates were analyzed by SDS/PAGE and sequential blotting with antibodies against EGF receptor, Alix and HCRP1. The same membrane was then reblotted with anti-tubulin to verify equal loadings. The experiment was repeated three times with the same result.

View larger version (107K): [in a new window]   Fig. 7. Alix depletion affects LBPA but not CD63 levels in multivesicular endosomes. Subcellular localisation of LBPA and CD63 in control (A,C) and Alix-siRNA-treated (B,D) cells. Cells were labelled with monoclonal antibodies against LBPA and CD63 followed by a rabbit anti-mouse antibody and 15 nm proteinA gold. In control cells, LBPA was found in typical MVBs and multilamellar late endosomes. Less staining was observed in Alix-siRNA-treated cells. CD63 labelling was also found in MVBs and late endosomes. Quantitation of the labelling intensity (E,F) indicated that total cell and endosomal LBPA content was significantly reduced, whereas CD63 levels were close to control values. Scale bars: 200 nm.

View larger version (78K): [in a new window]   Fig. 8. Accumulation of abnormal cortical actin structures in response to lowered Alix levels. HeLa cells treated with control RNA (A-C) or with siRNA (D-F) against Alix (see Fig. 1B) were stained with rhodamine-phalloidin (B-E), anti-cortactin (C-F) and anti-EEA1 (A-D). Yellow shows colocalisation between actin and cortactin in abnormal actin structures. Scale bars: 5 µm.

View larger version (90K): [in a new window]   Fig. 9. The F-actin structures that accumulate in Alix-depleted cells colocalise with clathrin. HeLa cells treated with control RNA (A,B) or with siRNA (C,D) against Alix (see Fig. 1B) were stained with rhodamine-phalloidin (A-C) and anti-clathrin (B-D). Yellow indicates colocalisation between actin and clathrin. Scale bars: 5 µm.

View larger version (106K): [in a new window]   Fig. 10. The F-actin structures that accumulate in Alix-depleted cells are not accessible to fluid phase markers. HeLa cells were treated with control RNA (A) or with siRNA (B) against Alix (see Fig. 1B). The cells were left to internalise biotinylated dextran (5 mg/ml) for 30 minutes at 37°C, and then washed once with medium at 37°C. The cells were permeabilised before fixation on ice and stained for confocal microscopy using FITC-streptavidin (green) and rhodamine-phalloidin (red). Scale bars: 5 µm.

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