Loss of RAB7 slows EGFR degradation. A, HeLa cells were transfected with no siRNA (-), 100 nm siCON, or RAB7 siRNA at a concentration of 100, 25, 10, and 1 nm (lanes 2–5, respectively). Total cell lysate (20 μg) was resolved by 12% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with the indicated antibodies. B, HeLa cells were transfected with no siRNA (-), RAB7 siRNA for 24, 48, and 72 h, or siCON for 72 h, as indicated. Total cell lysate (20 μg) was resolved by 12% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with the indicated antibodies. C and D, HeLa cells were transfected with nothing (untransfected), siCON, or RAB7 siRNA and allowed to recover for 72 h. ¹²⁵I-EGF degradation and ¹²⁵I secretion were monitored as described under “Materials and Methods.” Data are plotted as the percentage of intact total radioactivity (B) and the percentage of secreted total radioactivity (C). n = 3; mean ± S.E. ^**, p < 0.01 (Student's t test) as compared with either siCON-transfected or untransfected samples.

Loss of RAB7 does not affect EGFR progression through EEA1 positive early endosomes. A, HeLa cells were transfected with nothing (No siRNA), RAB7 siRNA, or siCON and grown on coverslips. Seventy-two hours post-transfection, the cells were serum-starved for 2 h and pulse-labeled with Texas Red-EGF (TR-EGF) (Molecular Probes) as described under “Materials and Methods.” At the indicated time points, the cells were fixed and processed for confocal immunofluorescence microscopy using an anti-EEA1 mouse monoclonal antibody (Transduction Laboratories). Images were collected with an Olympus FV500 confocal microscope and processed using the FV500 software. Insets are higher resolution images of the boxed region. Images are single planes and representative of three independent experiments. Size bar, 10 μm. Inset size bar, 1 μm. B, endosomes positive for both EEA1 and Texas Red EGF were counted and plotted as a percentage of total EEA1-positive endosomes. Data are plotted as the mean ± S.E. (n = 3). Three consecutive planes (0.20-μm steps) from three separate fields containing 5–15 cells were analyzed from each experiment for each condition. ^*, p < 0.05 (Student's t test). Control (C) or RAB7 siRNA-transfected cells (D) were pulse-labeled with ¹²⁵I-EGF for 15 min at 37 °C and chased with DMEM for an additional 45 min at 37 °C. Postnuclear supernatant from these cells was separated over a 17% Percoll gradient. Fractions were collected and assayed for associated radioactivity. Data are plotted as the percentage of total radioactivity found in each fraction. An immunoblot was performed on every other fraction using LAMP-1 to mark late endosomes/lysosomes and EEA1 to mark early endosomes. Diamonds on the x axis represent migration of density beads (∼R_f0.22 = 1.040 g/ml; ∼R_f0.8 = 1.069 g/ml; ∼R_f0.85 = 1.080 g/ml; ∼R_f0.9 = 1.109 g/ml). WT, wild type.

Loss of RAB7 results in the accumulation of EGF in CD63-positive endosomes. A, HeLa cells were transfected with nothing (No siRNA), RAB7 siRNA, or siCON and pulse-labeled with Texas Red-EGF (TR-EGF) (red) for the indicated times. Cells were fixed and processed for indirect immunofluorescence staining with antibody against CD63 (multivesicular bodies) (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Size bar, 10 μm. Images were collected with an Olympus FV1000 confocal microscope and processed using the FV1000 software. Insets are higher resolution images of the boxed region. Top, Texas Red signal; middle, CD63 signal; bottom, merged image. Inset size bar, 1 μm. Shown are single planes that are representative of 3–6 independent experiments. B, endosomes positive for both CD63 and Texas Red EGF were counted and plotted as a percentage of total CD63-positive endosomes. Data are plotted as the mean ± S.E. (n = 3, 10 min; n = 6, 30, 60, and 90 min). Three consecutive planes from three separate fields containing 5–15 cells were analyzed from each experiment for each condition. ^***, p ≤ 0.001 (Student's t test).

Total lysosomal enzyme activity is decreased and endocytic compartment density is altered in the absence of RAB7. A, HeLa cells were transfected with RAB7 siRNA or siCON. Seventy-two hours post-transfection, cells were stimulated without (0′ EGF) or with EGF (60′ EGF) for 60 min. PNS was collected from an equal number of cells and assayed for total β-galactosidase activity. Enzyme activity was normalized to protein levels. Data were normalized to enzyme activity from unstimulated siCON-treated cells. Data are shown as the mean ± S.E. from five independent experiments. ^*, p < 0.05 (Student's t test). siCON-transfected (B) and RAB7 siRNA-transfected HeLa cells (C) were stimulated for 60 min with EGF. PNS was separated over a 17% Percoll gradient, and the late endosomal fractions (R_f 1.0-0.7) were then further resolved over a 28% Percoll gradient. Fractions from the 28% gradient were assayed for β-galactosidase activity, and each fraction was plotted as a percentage of total activity. Data are representative of four independent experiments. Diamonds on the x axis represent migration of density beads (R_f0.2 = 1.040 g/ml; R_f0.48 = 1.055 g/ml; R_f0.74 = 1.069 g/ml; R_f0.79 = 1.080 g/ml; R_f0.85 = 1.109 g/ml). Two consecutive fractions from the first 26 fractions of the 28% Percoll gradient were combined, resuspended in 2× Sample Buffer, resolved by 7.5% SDS-PAGE, and immunoblotted (IB) for the presence of CD63 (insets).

Rab7 is not required for the biogenesis of LE/MVBs or ILV formation, but loss of RAB7 alters LE/MVB size and morphology. HeLa cells were transfected with siCON or RAB7 siRNA. After 72 h of recovery, cells were stimulated for 60 min with EGF and processed for electron microscopy. Shown are representative electron micrographs of sections from siCON-treated (A) and RAB7 siRNA-treated cells (B). Endocytic compartments are marked as follows: LE/MVBs (large arrowheads), clustered lysosomes (small arrowhead), or aberrant compartments containing intraluminal vesicles (arrows). Scale bars, 2 μm. C–G, representative LE/MVBs and lysosomes are shown for each condition. Scale bar, 500 nm.

EGFR is sequestered in intraluminal vesicles in the absence of RAB7. A, HeLa cells were transfected with RAB7 siRNA or siCON. After 72 h of recovery, cells were stimulated with EGF for 5, 30, or 60 min. Cells were digitonin-permeabilized, and membranes were isolated and incubated with nothing or with proteinase K in the presence or absence of Triton X-100. The samples were resolved by 7.5% SDS-PAGE and immunoblotted for EGFR. Shown is a blot from a representative experiment (n = 6). B, quantification of EGFR degradation. EGFR levels (without proteinase K or Triton X-100) from A were measured using ImageJ (National Institutes of Health) and plotted as a percentage of total EGFR at 5 min. C, quantification of protease resistance. EGFR levels (with proteinase K treatment only) from A were measured using ImageJ and plotted as a percentage of total EGFR at 5 min. Data are plotted as the mean ± S.E. (n = 6). ^*, p < 0.05 (Student's t test).

Rescue of RAB7 knockdown. A, HeLa cells were transfected with RAB7-specific siRNA or control siRNA. Forty-eight hours after transfection, the cells were calcium phosphate-transfected with wild type, HA-tagged canine Rab7 (canine HA-Rab7). 24 h later, cells were serum-starved for 2 h, followed by stimulation with 10 ng/ml EGF for 0, 15, or 60 min. Cell lysates were prepared, resolved by either 7.5% SDS-PAGE (EGFR) or 12% SDS-PAGE (RAB7, α-tubulin), and immunoblotted (IB) as indicated. B, tTA-HeLa cells were transfected with RAB7-specific siRNA or siCON. Forty-eight hours after transfection, cells were infected with nothing, tetracycline-regulatable adenovirus encoding for the HA-tagged, constitutively active mutant of canine Rab7 (HA-Rab7(Q67L)), or tetracycline-regulatable adenovirus encoding for the HA-tagged, dominant negative mutant of canine Rab7 (HA-Rab7(N125I)). Cells were also infected with HA-Rab7(Q67L) in the presence of tetracycline to inhibit protein expression. After recovery from infection for 24 h, cells were assayed for ¹²⁵I-EGF degradation following incubation with ¹²⁵I-EGF for 60 min. Data are plotted as the percentage of intact ¹²⁵I-EGF (n = 3; mean ± S.E.). ^*, p < 0.05; ^**, p < 0.01 (Student's t test) as compared with untransfected and siCON controls. C, cell lysates were prepared from cells treated as in B, resolved by 12% SDS-PAGE, and immunoblotted with an anti-RAB7 antibody for the presence of endogenous Rab7 and exogenous HA-Rab7(Q67L) or HA-Rab7(N125I).

Knockdown of RAB7 does not affect transferrin recycling through the early endosome. HeLa cells were transfected with nothing or siCON or RAB7 siRNA. After recovery, cells were analyzed for ¹²⁵I-Tfn recycling, as described under “Materials and Methods.” As a positive control to inhibit ¹²⁵I-Tfn recycling, cells were incubated with 100 μm monensin. A, time course of internalized ¹²⁵I-Tfn; B, time course of secreted ¹²⁵I-Tfn. Data are plotted as the mean ± S.E. (n = 3). ^*, p < 0.05; ^**, p < 0.01 (Student's t test).

The EGFR does not reach LAMP-2-positive lysosomes in the absence of RAB7. A, HeLa cells were transfected with nothing (No siRNA), RAB7 siRNA, or siCON and pulse-labeled with Texas Red EGF (red) for the indicated times. Cells were fixed and processed for indirect immunofluorescence staining with antibody against LAMP-2 (lysosomes) (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Size bar, 10 μm. Images were collected with an Olympus FV1000 confocal microscope and processed using the FV1000 software. Insets are higher resolution images of the boxed region. Top, Texas Red signal; middle, CD63 signal; bottom, merged image. Inset size bar, 1 μm. Shown are single planes that are representative of three independent experiments. B, endosomes positive for both LAMP-2 and Texas Red EGF were counted and plotted as a percentage of total endosomes positive for LAMP-2 only. Data are plotted as the mean ± S.E. (n = 3). Three consecutive planes from three separate fields containing 5–15 cells were analyzed from each experiment for each condition. ^*, p < 0.05 (Student's t test).

¹²⁵I-EGF accumulates in multiple high density fractions in the absence of RAB7. HeLa cells transfected with RAB7 siRNA were pulse-labeled with ¹²⁵I-EGF for 15 min and chased in DMEM for 45 min at 37 °C. PNS from these cells was resolved over consecutive 17 and 28% Percoll gradients as in Fig. 6B. Fractions from the 28% gradient were collected and assayed for both radioactivity and β-galactosidase activity (β-gal). Data are plotted as a percentage of total enzyme activity (triangle) or radioactivity (square). Data are representative of three independent experiments. The diamonds represent the density bead markers described in the legend to Fig. 6.

Depletion of RAB7 results in the accumulation of enlarged late endocytic compartments. A, size distribution of maximum diameter (nm) for MVBs (siCON, n = 282; RAB7 siRNA, n = 387). B, size distribution (nm) of maximum diameter for lysosomes (siCON, n = 259; RAB7 siRNA, n = 151). C, percentage of total cytosolic area occupied by characteristic late endocytic compartments (mean ± S.E.). D, comparison of the average diameter (nm) of the late endocytic compartments observed in both conditions (mean ± S.E.). Total cytoplasmic area was as follows: siCON, 4914 μm²; RAB7 siRNA, 5000 μm². Data are representative of 105 sections/condition analyzed from three independent experiments. ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001 (Student's t test). E and F, ILVs were counted from 75 MVBs (25 MVBs/experiment; n = 3) and plotted as the average number of ILVs/MVB (E) or the mean number of ILVs/MVB (F). Data are plotted as the mean ± S.E. ^***, p ≤ 0.001 (Student's t test).

Model for endocytic defects in the absence of RAB7. In wild-type HeLa cells, the EGFR is internalized via clathrin-coated pits from the plasma membrane, sorted through early endosomes to LE/MVBs, and delivered to lysosomes where it is degraded. LE/MVBs mature from early endosomes as cargo is sequestered within intraluminal vesicles. Based on our data in RAB7 knockdown cells, RAB7 is only needed to move cargo from the LE/MVB to lysosomes. Loss of RAB7 does not affect movement of the EGF·EGFR complex through the early endosome or the formation of ILVs in the MVB. Loss of RAB7 causes the accumulation of densely packed, enlarged MVBs, with a concomitant decrease in lysosome number.