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First published online 29 January 2003
doi: 10.1242/jcs.00327

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Distinct caveolae-mediated endocytic pathways target the Golgi apparatus and the endoplasmic reticulum

Department of Pathology and Cell Biology, Université de Montréal, Montréal, Québec, Canada

View larger version (59K): [in a new window]   Fig. 1. DynK44A inhibits CTX internalization into endosomes and the Golgi apparatus. FITC-CTX internalized for 30 minutes at 37°C (A,D) is localized to both endosomes labeled for TfR (B) and the Golgi apparatus labeled for GM130 (E). The merged confocal images present FITC-CTX in green (C,F) and TfR (C) or GM130 (F) in red and colocalization in yellow. Alternatively, NIH-3T3 cells were infected with both the tTA and dynK44A adenoviruses (G-J), and after 36 hours, the cells were coincubated with FITC-CTX (H) and Rh-Tf (I) for 30 minutes at 37°C prior to fixation. HA-tagged dynK44A-infected cells (arrows) were identified by postfixation labeling with anti-HA antibodies followed by Alexa647 anti-mouse secondary antibodies (G). The merged confocal image presents FITC-CTX in green, Rh-Tf in red and colocalization in yellow (J). Dramatic reduction in CTX and Rh-Tf internalization was observed in the dynK44A expressing cell. Bar, 20 µm.

View larger version (23K): [in a new window]   Fig. 2. Caveolae mediate CTX endocytosis. FITC-CTX (empty bars) and Rh-Tf (filled bars) were endocytosed for 30 minutes at 37°C in cells infected with wild-type dynamin, dynK44A, clathrin hub and caveolin-1 adenoviruses. Endocytosis into the perinuclear region was quantified in uninfected cells and in infected cells identified (as indicated) by postfixation labeling for the appropriate epitope marker. The degree of endocytosis is presented as the percentage of fluorescence intensity relative to uninfected control cells (A). Cell-surface FITC-CTX binding at 4°C was quantified in the adenovirus-infected cells and is presented as the percentage of fluorescence intensity relative to uninfected control cells (B). The data represent the average of three different experiments (±s.e.m.). The ability of dynK44A, but not the clathrin hub, to inhibit CTX endocytosis together with its reduction in caveolin-1 infected cells demonstrates the existence of a caveolae-mediated CTX endocytic pathway.

View larger version (86K): [in a new window]   Fig. 3. MßCD blocks CTX delivery to the Golgi but not to endosomes. NIH-3T3 cells were either left untreated (A-C) or pretreated with 5 mM of mßCD (D-L) for 30 minutes then pulse labeled with FITC-CTX (A,D,G) and Rh-Tf (B,E,H) for 30 minutes at 37°C. Images presenting FITC-CTX internalization in the absence or presence of mßCD (A,D) were obtained using the same acquisition parameters. A smaller pinhole (0.6 Airy units) and increased zoom (G-L) were used to assess the overlap between internalized FITC-CTX (G), Rh-Tf-labeled endosomes (H) and the GM130-labeled Golgi apparatus (I). Merged confocal images present FITC-CTX in green and either Rh-Tf in red (C,F,K) or GM130 in red (L) and colocalization in yellow. A triple merge shows FITC-CTX in green, Rh-Tf in red and GM130 in blue (J). In mßCD-treated cells, cell-surface FITC-CTX labeling is significantly increased, and CTX is still internalized with transferrin to endosomes but not to the Golgi. Bars: A-F in F, 20 µm; G-L in L, 8 µm.

View larger version (71K): [in a new window]   Fig. 4. Caveolin-1 overexpression negatively regulates CTX internalization to the Golgi apparatus but not to endosomes. NIH-3T3 cells were infected with the tTA and caveolin-1 adenoviruses and after 36 hours pulse labeled with FITC-CTX (A) for 30 minutes at 37°C prior to fixation. Cells were then triple labeled with anti-caveolin antibodies and Alexa 568 anti-rabbit antibodies (B) and anti-GM130 and Alexa 647 anti-mouse antibodies (C). The infected cell is indicated by an arrow, and the merged confocal image (FITC-CTX in green and GM130 in red) shows reduced FITC-CTX internalization to the Golgi in the caveolin-1-infected cell (D). Increased zoom of a caveolin-1-overexpressing cell (E-J) shows internalized FITC-CTX (E) and postfixation caveolin (F) and TfR (G) labeling. Merged confocal images present TfR in green and caveolin in red (H) and FITC-CTX in green and either caveolin (I) or TfR (J) in red. Quantification by mask overlay of FITC-CTX internalization to the GM130-positive Golgi (K) or to TfR-positive endosomes (L) shows that in cells overexpressing caveolin-1, FITC-CTX delivery to the Golgi apparatus is reduced. Bars: A-D in D, 20 µm; E-J in J, 8 µm.

View larger version (56K): [in a new window]   Fig. 5. CTX endocytosis in clathrin-hub-expressing cells. NIH-3T3 cells infected for 36 hours with the tTA and clathrin hub adenoviruses were incubated at 37°C with FITC-CTX for 5 (A-D) or 30 minutes (E-H). The distribution of FITC-CTX was visualized directly (A,E) and post-fixation labeling with anti-caveolin (B,F) and anti-GM130 (C,G) antibodies revealed with the appropriate Alexa-568- and Alexa-647-conjugated secondary antibodies, respectively. Merged confocal images (D,H) present FITC-CTX in green, caveolin in red and GM130 in blue. After five minutes of endocytosis, CTX is primarily associated with caveolae and with time accumulates in the perinuclear region, where, after 30 minutes, it colocalizes extensively with the Golgi apparatus. Bar, 8 µm.

View larger version (46K): [in a new window]   Fig. 6. Caveolin-1 overexpression reduces delivery of AMF-FITC to the smooth ER. NIH-3T3 cells were infected with the tTA and caveolin-1 adenoviruses and after 36 hours pulse labeled with AMF-FITC for 60 minutes at 37°C prior to fixation. AMF-FITC was revealed with rabbit anti-FITC followed by Alexa 488 anti-rabbit antibodies (A) and the smooth ER labeled with anti-AMF-R mAb followed by Alexa 647-anti-rat IgM antibodies (B). The caveolin-1-overexpressing cell (arrow) was detected with anti-c-Myc antibodies followed by Texas-Red anti-mouse antibodies (C). The merged confocal image presents AMF-FITC in green, the AMF-R-labeled smooth ER in red and colocalization in yellow (D). Quantification of AMF endocytosis to AMF-R-positive smooth ER tubules (E) shows that caveolin-1-expressing cells exhibit a significant decrease in the caveolae-mediated endocytosis of AMF. Bar, 20 µm.

View larger version (134K): [in a new window]   Fig. 7. BFA treatment inhibits the caveolae-mediated endocytosis of CTX to the Golgi apparatus but not of AMF to the smooth ER. NIH-3T3 cells were pretreated with 10 µg/ml BFA for 30 minutes at 37°C and then incubated in the presence of BFA with 5 µg/ml FITC-CTX for 30 minutes at 37°C (A-F) or with 50 µg/ml AMF-FITC for 60 minutes at 37°C (G-I). Cells incubated with FITC-CTX were labeled with either anti-GM130 (B) or anti-TfR (E) antibodies and merged confocal images present FITC-CTX in green and GM130 (C) or TfR (F) in red and colocalization in yellow. Internalized AMF-FITC was revealed with rabbit anti-FITC (G) and AMF-R tubules with anti-AMF-R mAb (H) followed by the appropriate secondary antibodies. The merged confocal image (I) presents the AMF in green and AMF-R in red and colocalization in yellow. In the presence of BFA, CTX is delivered to the endosomes but not to Golgi fragments, whereas AMF is still delivered to AMF-R-labeled smooth ER (arrows). Bar, 8 µm.

View larger version (144K): [in a new window]   Fig. 8. Nocodazole treatment and a 20°C temperature block inhibit the caveolae-mediated endocytosis of CTX to the Golgi but not of AMF to the smooth ER. NIH-3T3 fibroblasts pretreated with 10 µM nocodazole at 37°C (A-K) were pulse labeled with either 5 µg/ml FITC-CTX for 30 minutes at 37°C (A-H) or with 50 µg/ml AMF-FITC for 60 minutes at 37°C (I-K) in the presence of nocodazole. Alternatively, cells were incubated with 50 µg/ml AMF-FITC for 60 minutes at 20°C (L-N). Overlap of endocytosed FITC-CTX (A,D) with anti-GM130 (B), anti-caveolin (E) or anti-TfR (F) labeling and of AMF-FITC (I, L) with anti-AMF-R (J, M) labeling was determined. The merged confocal images (C,G,H,K,N) present the indicated labels in red and green and colocalization in yellow. In the presence of nocodazole FITC-CTX is not delivered to the fragmented Golgi (A-C) but remains associated with caveolin and TfR-positive endosomes (D-H). Neither nocodazole treatment (I-K) nor a 20°C temperature block (L-N) prevent AMF delivery to the smooth ER (arrows). Bar, 8 µm.

View larger version (129K): [in a new window]   Fig. 9. AMF and CTX do not cointernalize. NIH-3T3 cells were coincubated with 50 µg/ml of AMF-FITC and 5 µg/ml of Alexa 594-CTX at 37°C for 5 minutes (A-F) or 30 minutes (G-J) prior to fixation with precooled methanol/acetone. The distributions of AMF-FITC, revealed with rabbit anti-FITC followed by Alexa 488 anti-rabbit antibodies (A and G), and of Alexa-594–CTX (B, H) were compared after five minutes with the smooth ER labeled with anti-AMF-R mAb and Alexa 647 anti-rat IgM antibodies (C) or at 30 minutes with the Golgi apparatus labeled with anti-GM130 mAb and Alexa 647 anti-mouse antibodies (I). Merged confocal images present AMF-FITC in green and either AMF-R (D) or Alexa 594-CTX (E) in red. Triple merges show AMF-FITC in green, Alexa 594-CTX in red and either AMF-R (F) or GM130 (J) in blue. Bar, 8 µm.

View larger version (99K): [in a new window]   Fig. 10. Targeting of AMF to the ER and CTX to the Golgi apparatus is tyrosine kinase dependent. NIH-3T3 cells were pretreated with genistein (100 µg/ml) for 30 minutes during ligand incubation (A-I) or left untreated (J-L). Cells were pulse labeled with FITC-CTX for 30 minutes at 37°C (A,D) and labeled with monoclonal anti-GM130 (B) or anti-TfR antibodies (F) following by Alexa 568 anti-mouse secondary antibody. The merged confocal images present FITC-CTX in green and either GM130 (C) or TfR (F) in red and colocalization in yellow (C,F). Genistein selectively inhibits CTX delivery to the Golgi but not to TfR-positive endosomes. Alternatively, NIH-3T3 cells were pulse labeled with AMF-FITC for 5 minutes at 37°C. AMF-FITC was revealed with rabbit anti-FITC followed by Alexa 488 anti-rabbit antibodies (G,J) and the smooth ER labeled with anti-AMF-R followed by rhodamine-red-X anti-rat IgM antibodies (H, K). The merged confocal images present AMF-FITC in green and AMF-R in red and colocalization in yellow (I,L). The image of AMF endocytosis in the presence of genistein (G) was acquired at the same intensity level as the control in the absence of genistein (J), clearly demonstrating that genistein inhibits AMF delivery to the smooth ER. Bar, 8 µm.

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