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First published online October 24, 2007
doi: 10.1242/10.1242/jcs.017301

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RILP is required for the proper morphology and function of late endosomes

Cinzia Progida^1,*, Lene Malerød^2,*, Susanne Stuffers², Andreas Brech², Cecilia Bucci^1, and Harald Stenmark^2,

¹ Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Via Provinciale Monteroni, 73100 Lecce, Italy
² Department of Biochemistry, Institute for Cancer Research, the Norwegian Radium Hospital and Centre for Cancer Biomedicine, University of Oslo, Montebello, N-0310 Oslo, Norway

View larger version (47K): [in this window] [in a new window]   Fig. 1. Depletion of RILP is accompanied by depletion of Vps22, and vice versa. HeLa cells were treated with control RNA, siRNA against RILP, siRNA against Vps22 or both siRNAs against RILP and Vps22. Cell lysates were analyzed by western blotting against Vps22, RILP or -tubulin (serving as a loading control).

View larger version (36K): [in this window] [in a new window]   Fig. 2. Depletion of RILP inhibits ligand-induced EGFR degradation as studied by western blotting. (A) HeLa cells treated with control RNA or siRNA against RILP or Vps22 were stimulated for 15, 60, 120 or 180 minutes with 50 ng/ml EGF in the presence of 10 µg/ml cycloheximide before the cells were lysed and analyzed by SDS-PAGE and western blotting with antibodies against EGFR. The same membrane was then reblotted with antibody against -tubulin to verify equal loadings. (B) The intensities of the EGFR staining were quantified and plotted as a percentage of the respective intensities after 15 minutes of EGF stimulation. The error bars represent the s.e.m. of three experiments. According to a Student's t-test, P<0.05 for the 120 and 180 minutes time points.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Depletion of RILP inhibits ligand-induced EGFR degradation as studied by confocal microscopy. HeLa cells treated with control RNA (`A'), siRNA against RILP (`B') or siRNA against Vps22 (`C') were pretreated with 10 µg/ml cycloheximide (1 hour) before they were stimulated for 15, 60, 120 or 180 minutes with 50 ng/ml EGF. (A) The cells were permeabilized before fixation and stained for EGFR (red). (B) The intensities of the EGFR staining were quantified using Zeiss LSM 510 software (version 3.2) and plotted as a percentage of the respective intensities after 15 minutes of EGF stimulation. The error bars represent the s.e.m. of three experiments. According to Student's t-test, significantly less EGFR was degraded in RILP- or Vps22-depleted cells compared with control RNA cells (P<0.05 for all time points).

View larger version (75K): [in this window] [in a new window]   Fig. 4. Depletion of RILP retards internalized EGFRs in early endosomes. HeLa cells transfected with control RNA or RILP siRNA were pretreated with 10 µg/ml cycloheximide (1 hour) and stimulated with 50 ng/ml EGF for 2 hours. The cells were permeabilized before fixation and stained with antibodies against EGFR (red), Lamp1 (green) and EEA1 (blue).

View larger version (39K): [in this window] [in a new window]   Fig. 5. Depletion of RILP decreases lysosomal degradation of CI-M6PR without affecting its trafficking from the TGN to endosomes. (A) The expression of CI-M6PR (red), TGN46 (green) and EEA1 (blue) was determined in HeLa cells treated with control RNA or siRNA against RILP by confocal immunofluorescence. (B) All the images were scanned at the same settings below saturation, and the intensities of CI-M6PR, TGN46 and EEA1 (relative to the control) were determined by the Zeiss LSM 510 software (version 3.2). The graph represents the average (±s.e.) of three individual experiments, where at least 15 cells were quantified per experiment. (C) The expression of CI-M6PR in control cells and RILP siRNA cells was determined by western blotting, using -tubulin as a loading control. (D) CI-M6PR expression (relative to -tubulin) was quantified from three separate western blot experiments (±s.e.). The quantifications in B and D showed significantly higher levels of CI-M6PR in RILP-depleted cells than in control cells according to a Student's t-test (P<0.05).

View larger version (16K): [in this window] [in a new window]   Fig. 6. Depletion of RILP or Vps22 does not affect transferrin trafficking through early endosomes. HeLa cells treated with control RNA (scr), with siRNA against RILP or with siRNA against Vps22 were allowed to bind to 125I-labelled human transferrin (hTf) for 1 hour at 0°C. After washing with cold PBS-BSA to remove the unbound hTf, cells were incubated for different times at 37°C to allow internalization of the surface-bound 125I-labelled hTf. Internal 125I-labelled hTf was estimated as the residual radioactivity after pronase treatment, compared with total bound 125I-labelled hTf. Bars, s.e. of triplicate samples.

View larger version (63K): [in this window] [in a new window]   Fig. 7. Increased LBPA and Lamp1 staining in RILP-depleted cells. (A) HeLa cells treated with control RNA (upper panels) or siRNA against RILP (lower panels) were fixed and stained with antibodies against LBPA (red), Lamp1 (green) and EEA1 (blue) and studied by confocal microscopy under identical settings. Merged images are shown in the right-hand panels. Note the increased staining of LBPA and Lamp1 in the RILP-depleted cells. (B) Quantification of results from confocal microscopy (20 cells from two different experiments) showed significantly increased LBPA and Lamp1 intensities in RILP-depleted cells compared with that of control cells (P<0.05 according to Student's t-test).

View larger version (127K): [in this window] [in a new window]   Fig. 8. Depletion of RILP alters the morphology of late endosomes. (A) In control cells, CD63 labelling is typically found in MVEs with varying numbers of intralumenal vesicles. Arrowheads indicate MVBs. (B) Cells depleted for RILP frequently contain more clustered CD63-positive endosomes. These endosomes are mostly devoid of intralumenal vesicles in comparison with controls. CD63 is labelled with 10-nm protein-A-gold; bars, 200 nm.