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First published online 24 June 2008
doi: 10.1242/jcs.014977

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Caveolin-1-dependent β1 integrin endocytosis is a critical regulator of fibronectin turnover

Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, 211 Bailey Road, West Henrietta, NY 14586, USA

View larger version (95K): [in this window] [in a new window]   Fig. 1. Colocalization of internalized fibronectin with β1 integrins. (A-C) FN-null myofibroblasts were incubated with 10 µg/ml TR-fibronectin overnight. Cells were washed and then incubated for 12 hours in cell culture media lacking fibronectin, but containing 50 µM chloroquine. Cells were stained with an anti-β1 integrin antibody (HMβ1-1). (A) TR-fibronectin; (B) β1 integrin; (C) overlay image. (D-F) FN-null myofibroblasts were incubated with 10 µg/ml TR-fibronectin overnight. Cells were washed, and then incubated with 10 µg/ml 9EG7 at 4°C for 30 minutes. Cells were washed, and then chased for 4 hours with cell culture medium lacking fibronectin, but containing 100 µM chloroquine. Cells were then stained with a FITC-conjugated secondary antibody. (D) TR-fibronectin; (E) 9EG7 (β1 integrin); (F) overlay image. (G-I) Smooth muscle cells were incubated with 10 µg/ml TR-fibronectin and 50 µM chloroquine for 12 hours. Cells were stained using an anti-β1 integrin antibody. (G) TR-fibronectin; (H) β1 integrin (FITC); (I) overlay image. All images are optical sections collected from a confocal microscope. Scale bars: 10 µm.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Colocalization of internalized fibronectin and β1 integrins shortly after initiation of endocytosis. FN-null myofibroblasts were incubated with 10 µg/ml TR-fibronectin and 50 µg/ml FITC-conjugated 9EG7 at 4°C for 1 hour during the pulse. After 30 minutes of chase at 37°C, cells were incubated with 0.2% Trypan Blue for 3 minutes to quench extracellular fluorescence. (A) TR-fibronectin; (B) FITC-conjugated 9EG7; (C) overlay images. Arrowheads in C indicate colocalized TR-Fibronectin and FITC-9EG7. These images are optical sections collected from a confocal microscope. Scale bar: 10 µm.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Colocalization of internalized fibronectin with 5 integrins. Smooth muscle cells were incubated with 10 µg/ml TR-fibronectin and 50 µM chloroquine for 8 hours. Cells were stained using an anti-5 integrin antibody (AB1928). (A) TR-fibronectin; (B) 5 integrin; (C) overlay image. These images are optical sections collected from a confocal microscope. Scale bar: 10 µm.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Quantification of fibronectin endocytosis. (A) FN-null myofibroblasts were incubated with 2.5-50 µg/ml AF488-conjugated fibronectin at 4°C during the pulse phase (total endocytosis). Some samples were co-incubated with 800 µg/ml unlabeled fibronectin to determine non-specific endocytosis. Cells were chased in the absence of fibronectin for 2 hours at 37°C, and then processed for flow cytometry to quantify endocytosed fibronectin. Specific endocytosis was determined by subtracting nonspecific endocytosis from total endocytosis. The graph shows the best-fit curve of the mean fluorescence intensity (MFI) of internalized AF488-fibronectin. Data represent the mean of duplicate samples and error bars the range. (B) FN-null myofibroblasts were incubated with 10 µg/ml AF488-fibronectin in the presence of increasing concentrations of unlabeled fibronectin (0-800 µg/ml) during the pulse phase. Cells were chased in the absence of fibronectin for 2 hours at 37°C, and then processed for flow cytometry to quantify endocytosed AF488-fibronectin. The amount of internalized AF488-fibronectin is reported as percent control internalization (cells incubated in the absence of unlabelled fibronectin, which was set equal to 100%). Data represent the mean of duplicate samples and error bars indicate the range.

View larger version (45K): [in this window] [in a new window]   Fig. 5. Blocking of β1 integrin inhibits fibronectin endocytosis. (A-C) FN-null myofibroblasts were incubated with 25 µg/ml β1 antibodies (Ha2/5) or isotype control antibodies at 4°C for 30 minutes. 10 µg/ml TR-fibronectin (A,B) or AF488-fibronectin (C) was then added to the medium and cells were processed for fibronectin endocytosis pulse-chase assays. After 2 hours of chase, cells were either fixed for imaging assay (A, β1 inhibition; B, isotype control), or processed for flow cytometry to quantify endocytosed fibronectin (C). The numbers over the peaks in C are the MFI of internalized AF 488-fibronectin. (D-F) Smooth muscle cells were seeded in serum-free medium on vitronectin-coated dishes. Cells were allowed to adhere for 3 hours and then processed for integrin-blocking assay as described above for FN-null myofibroblasts. (D) 25 µg/ml β1 inhibitory antibodies (Ha2/5); (E) isotype control; blue: DAPI. (F) Quantification of endocytosed fibronectin in smooth muscle cells by flow cytometry. The numbers over the peaks in F are the MFI of internalized AF 488-fibronectin. Scale bars: 10 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 6. Blocking of β1 integrins inhibits endocytosis of fibronectin from preassembled matrix. FN-null myofibroblasts were incubated with either 30 µg/ml β1 inhibitory antibody (Ha2/5) or isotype control in suspension at room temperature for 30 minutes prior to seeding on pre-assembled TR-(A,B) or AF488 (C,D) fibronectin matrix. Cells were cultured for 24 hours at 37°C, and were then either fixed for imaging assay (A, β1 inhibition; B, isotype control) or processed for flow cytometry to quantify internalized fibronectin (C,D). The numbers over the peaks in C are the MFI of internalized AF488-fibronectin. Graph in D shows fold change relative to the MFI of endocytosed AF488-fibronectin in cells treated with isotype control IgM, which was set equal to 1 (n=4, mean ± s.d.). Scale bar: 10 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 7. Reduced endocytosis and turnover of pre-assembled fibronectin matrix in β1-integrin-null cells. GD25 and GD25 β1 re-expressing cells were seeded on pre-assembled TR (A,B,D,E,F,G) or AF488 (C) fibronectin matrix and incubated for 24 hours at 37°C. (A,B) Cells were incubated with 0.2% Trypan Blue for 3 minutes and then fixed. Intracellular fibronectin vesicles are shown (A, GD25; B, GD25 β1). The insets show outlines of cells loaded with CellTracker Green. Scale bar: 10 µm. (C) Cells were processed for flow cytometry to quantify endocytosed fibronectin. The numbers over the peaks in C are the MFI of internalized AF 488-fibronectin. (D-G) Cells were fixed without Trypan Blue treatment to visualize fibronectin fibrils (D,E, GD25; F,G, GD25 β1; E and G are phase images of corresponding fields). Scale bar: 40 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 8. Blocking of 5 integrin partially inhibits fibronectin endocytosis. FN-null myofibroblasts were incubated with 50 µg/ml 5 inhibitory antibodies (5H10-27) or 100 µg/ml v inhibitory antibodies (H9.2B8) or isotype control IgG at 4°C for 30 minutes. 10 µg/ml TR-fibronectin (A-D) or AF488-fibronectin (E) was added to the medium. After 2 hours of chase, cells were either fixed for imaging analysis (A, 5 inhibition; B, isotype control of 5; C, v inhibition; D, isotype control for v), or processed for flow cytometry (E) to quantify internalized fibronectin. The numbers over the peaks in E are the MFI of internalized AF488-fibronectin. Scale bar: 10 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 9. (A-F) Caveolin-1 regulates short-term fibronectin endocytosis. Cells expressing caveolin-1 siRNA (shcav, A-C) or control siRNA (shluc, D-F) were incubated with 10 µg/ml AF488-fibronectin and 50 µg/ml TR-conjugated 9EG7 at 4°C for 1 hour during the pulse. After 30 minutes of chase at 37°C, cells were incubated with 0.2% Trypan Blue for 3 minutes to quench extracellular fluorescence. AF488-fibronectin, A,D; TR-9EG7, B,E; overlay images, C,F. Scale bar: 10 µm. (G) Flow cytometric analysis of endocytosed fibronectin. Cells expressing caveolin-1 siRNA (shcav) or control siRNA (shluc) were incubated with 10 µg/ml AF488-fibronectin at 4°C for 1 hour in the presence or absence of 1 mg/ml unlabeled fibronectin. After 30 minutes of chase, cells were processed for flow cytometry. The specific MFI was determined by subtracting the total MFI (shcav, 315; shluc, 489) from the MFI of cells incubated with unlabeled fibronectin (shcav, 147; shluc, 177). Data show the fold change relative to the MFI of endocytosed AF488-fibronectin in control cells, which was set equal to 1 (mean ± range). (H-J) Colocalization of internalized fibronectin with lipid raft marker. FN-null myofibroblasts were incubated with 10 µg/ml TR-fibronectin and 2 µg/ml AF488-CTxB at 4°C for 1 hour. After 2 hours of chase, cells were incubated with 0.2% Trypan Blue for 3 minutes and then processed for imaging assay (H, TR-fibronectin; I, AF488-CTxB; J, overlay image, arrowheads point to colocalized TR-fibronectin and AF488-CTxB). (K-O) Colocalization of internalized fibronectin, β1 integrins and caveolin-1. FN-null myofibroblasts were incubated with 10 µg/ml TR-fibronectin overnight. Cells were washed and then incubated for 12 hours in cell culture media lacking fibronectin, but containing 50 µM chloroquine. Cells were stained with anti-β1-integrin and anti-caveolin-1 antibodies, followed by FITC-conjugated anti-hamster and Alexa Fluor 660-conjugated anti-rabbit secondary antibodies. (K, TR-fibronectin; L, β1 integrin; M, caveolin-1; N, overlay image, arrowheads show the colocalization of triple colors). Fluorescence intensity profiles (O) were generated using ImageJ software (National Institutes of Health, Bethesda, ML). A line was manually drawn to cross several TR-fibronectin containing vesicles (as shown in K) and the fluorescence intensity profiles were obtained from each individual color channel. The plot in O was generated in Excel (red, TR-Fibronectin; green, β1 integrin; blue, caveolin-1). All images are optical sections collected from a confocal microscope. Scale bar: 10 µm.

View larger version (56K): [in this window] [in a new window]   Fig. 10. Dominant-negative Eps15 does not inhibit fibronectin endocytosis. Dominant-negative Eps15 (A,C,E,G,I,K) or control Eps15 (B,D,F,H,J,L) were transiently expressed in FN-null myofibroblasts. (A-D) Transfected cells were seeded onto preassembled matrices and incubated for 24 hours. Endocytosed fibronectin is shown in A,B. (E-H) Short-term pulse-chase experiments were performed with transfected cells. (E,F) show endocytosed fibronectin following 2 hours of chase. (I-L) The effect of dominant-negative and control Eps15 on transferrin receptor endocytosis is shown. Transfected cells were detected by GFP expression (C,D,G,H,K,L) and manually outlined. 15-20 cells were analyzed for each condition; representative images are shown. Scale bar: 10 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 11. Integrin endocytosis in caveolin-1-knockdown cells. (A-F) Cells expressing caveolin-1 siRNA (shcav) or control cells (shluc) were incubated with 10 µg/ml TR-fibronectin overnight. Cells were washed, and then incubated for 8 hours in cell culture medium lacking fibronectin, but containing 50 µM chloroquine. Cells were stained with anti-β1 integrin antibody (FITC). Upper panels, shcav; lower panels, shluc. A,D, β1 integrin; B,E, TR-fibronectin; C,F, overlay images. (G-K) Cells expressing caveolin-1 siRNA (shcav, G,J) or control cells (shluc, H,K) were incubated with 50 µg/ml antibodies to 5 integrin (G-I) or β1 integrin (J,K) at 4°C for 45 minutes. Cells were then processed for integrin endocytosis assay. The fluorescence intensity of endocytosed 5 integrin (G,H) was quantified using a MATLab-based program. (I) Fold change relative to the fluorescence intensity of endocytosed 5 integrin in shluc cells, which was set equal to 1 (mean ± range from two independent experiments). All images are optical sections collected from a confocal microscope. Scale bars: 10 µm.

View larger version (56K): [in this window] [in a new window]   Fig. 12. Re-expression of caveolin-1 rescues endocytosis of β1 integrin in caveolin-1 siRNA cells. FN-null myofibroblasts expressing caveolin-1 siRNA (shcav) were transduced with Ad-cav or Ad-tet adenoviruses. (A-I) Cells were incubated with 10 µg/ml TR-fibronectin overnight. Cells were washed, and then incubated for 8 hours in cell culture medium lacking fibronectin, but containing 50 µM chloroquine. Cells were stained with anti-β1 integrin antibody (FITC). Upper panels, shcav without virus transduction; middle panels, Ad-cav; lower panels, Ad-tet. A,D,G: TR-fibronectin; B,E,H: β1 integrin; C,F,I, overlay images. Arrows in D show the internalized fibronectin; arrows in E show intracellular β1 integrins; arrows in F show colocalized fibronectin and β1 integrins. D`, E` and F` are enlarged images of area shown by the rectangle in F. (J-M) In the absence of fibronectin, cells were incubated with 50 µg/ml antibodies to 5 integrin (J) or β1 integrin (K-M) at 4°C for 45 minutes. Cells were then processed for integrin endocytosis assay. (J) The fluorescence intensity of endocytosed 5 integrin was quantified using a MATLab-based program. Graph shows the relative fluorescence intensity of endocytosed 5 integrin in shcav cells with or without virus transduction. Intracellular fluorescence intensity of shcav cells without virus transduction was set equal to 1 (mean ± range from two independent experiments). All images are optical sections collected by confocal microscopy. Scale bars: 20 µm.

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