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First published online 15 March 2005
doi: 10.1242/jcs.02280

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Internalization and recycling of ALCAM/CD166 detected by a fully human single-chain recombinant antibody

¹ Istituto Nazionale per la Ricerca sul Cancro, Largo R. Benzi 10, 16132 Genova, Italy
² Centro Biotecnologie Avanzate, Largo R. Benzi 10, 16132 Genova, Italy
³ Department of Experimental Oncology, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milano, Italy
⁴ Department of Experimental Pathology, University of Bologna, Via S. Giacomo 14, 40126 Bologna, Italy
⁵ Department of Experimental Medicine, University of Genova, Via L. B. Alberti 2, 16132 Genova, Italy

View larger version (67K): [in a new window]   Fig. 1. Characterization of the I/F8 antigen. (A) Immunoprecipitation from Sulfo-NHS-LC-biotin surface-labeled IGROV-I tumor cells using the I/F8 scFv coupled to CNBr-activated Sepharose beads. Immunoprecipitates were resolved by SDS-PAGE on a 10% polyacrylamide gel, and revealed by western blot analysis using HRP-conjugated Streptavidin and chemiluminescence. The I/F8 antigen is a 116 kDa monomer under both reducing (ME+) and non-reducing (ME–) conditions. Immunoprecipitates using an irrelevant scFv coupled to Sepharose beads were run as negative controls (ctr). (B) Silver-stained preparative immunoprecipitation and SDS-PAGE analysis of the I/F8 antigen. I/F8 immunoprecipitates from several tumor cell lines were resolved by SDS-PAGE on a 10% polyacrylamide gel under reducing conditions, and revealed by staining with silver nitrate. Bands were then excised from the gel and processed for MALDI-TOF analysis. (C) Immunofluorescence analysis of I/F8 (black line) and J4-81 (gray line) reactivity on various human and murine tumor cell lines. Broken line represents negative control with an irrelevant scFv. (D) Silver staining of I/F8 immunoprecipitated molecules from three murine tumor cell lines, resolved by SDS-PAGE on a 10% polyacrylamide gel. Numbers on left-hand side of blots indicate molecular weight markers in kDa.

View larger version (24K): [in a new window]   Fig. 2. Cell surface binding interference experiments between ALCAM/CD166 ligands and the I/F8 scFv, measured by flow cytometry on N592 SCLC cells. Ligand binding in the presence of I/F8 is indicated by black triangles and binding in the presence of the irrelevant anti-NIP scFv is indicated by empty triangles. CD6/Fc and ALCAM/Fc chimeras were used at 10 and 100 nM respectively.

View larger version (91K): [in a new window]   Fig. 3. I/F8-induced internalization of the ALCAM/CD166 molecule. IGROV-I cells were incubated with I/F8 scFv at 4°C in the presence (A,B,D) or in the absence (C) of the anti-FLAG-tag mAb as dimerizing agent. Cells were then placed on ice (A,B) or moved to 37°C for 30 minutes to induce internalization (C,D), and were subsequently untreated (A) or acid-washed to strip the surface-bound molecules (B-D). Samples were then fixed, permeabilized, stained with FITC-labeled secondary antibodies and visualized by using an Olympus confocal laser microscope system. Insets show staining with the irrelevant anti-NIP scFv as a negative control, except B where the Propidium Iodide-counterstained nuclei present in the same field are visualized. The residual staining detected in the stripped samples therefore corresponds to the endocytosed molecules. Magnification, 400 x.

View larger version (42K): [in a new window]   Fig. 4. Internalization kinetics of ALCAM/CD166 compared to transferrin. (A) ALCAM/CD166 internalization is time dependent and slower than transferrin. Confocal microscopic imaging of IGROV-I cells incubated with I/F8 scFv and anti-FLAG mAb in the presence of transferrin-FITC. The samples were allowed to internalize for the indicated times (in minutes) before acid-stripping of the surface-bound molecules, fixing, permeabilizing and staining with RITC-labeled secondary antibody. Images were acquired sequentially as single transcellular optical sections, and the same section is shown stained with I/F8 (left panels) and with transferrin (right panels). Insets show the non-stripped samples. (B) Internalized ALCAM/CD166 distributes in the sub-membrane region and does not cluster toward the perinuclear area. Confocal microscopic imaging of IGROV-I cells stained with I/F8 plus anti-FLAG mAb, allowed to internalize for 60 minutes, acid-washed to strip the surface if required (right panel), fixed, permeabilized and stained with FITC-labeled secondary antibody. Nuclei were counterstained with Propidium Iodide. Magnification, 600 x.

View larger version (136K): [in a new window]   Fig. 5. Internalized ALCAM/CD166 colocalizes primarily with clathrin. A2774 cells were stained either with I/F8 scFv plus anti-FLAG mAb (A,D) or with J4-81 mAb (B,E), allowed to internalize for 40 minutes at 37°C, acid-washed to remove the surface-bound antibody, fixed, permeabilized and stained with fluorochrome-labeled secondary antibody. As the colocalization with other molecules was investigated, the secondary antibody free valences were saturated by a 30-minute incubation with mouse IgG, followed by a second round of PFA fixation, and finally the cells were incubated with FITC-labeled anti-clathrin or with anti-caveolin antibodies. Anti-caveolin was then detected with RITC-labeled goat anti-mouse IgM antibody. As colocalizing positive controls, transferrin-RITC (TF, panel C) and cholera toxin B-FITC (CT B, panel F) were allowed to internalize for 10 and 40 minutes, respectively, at 37°C. Cells were then acid-washed, fixed, permeabilized and processed for staining with anti-clathrin and anti-caveolin antibodies. Double immunofluorescence was visualized by confocal microscopy. Single-color immunofluorescence images are shown in the small panels and the corresponding merged images are shown in the large panels. Original magnification, 600 x.

View larger version (104K): [in a new window]   Fig. 6. Ligand-triggered internalization of the ALCAM/CD166 molecule is maximal at the dividing cell contact region. Confocal microscopic imaging of A2774 cells stained with I/F8 plus anti-FLAG mAb, kept on ice (A,D) or allowed to internalize for 40 minutes (B,C), acid-washed to strip the surface if required (B,C,D), fixed, permeabilized and stained with FITC-labeled secondary antibody. Nuclei were counterstained with Propidium Iodide (original magnification 600x).

View larger version (43K): [in a new window]   Fig. 7. Ligand-induced internalization and recycling of ALCAM/CD166 molecules. (A) A2774 cells were surface-labeled with NHS-SS-biotin, incubated for 30 minutes at 37°C with or without the anti-ALCAM/CD166 mAb 3A6, treated with the reducing agent GSH, lysed and immunoprecipitated with I/F8 scFv coupled to Sepharose beads. Residual biotin-labeled molecules identify internalized proteins. (B) The kinetics of 3A6 mAb-triggered ALCAM/CD166 internalization was evaluated at different time points prior to GSH treatment and lysis. Optimal receptor internalization is at 40 minutes. The efficiency of surface NHS-SS-biotin stripping by GSH was evaluated at different time points on 3A6-treated cells maintained at 4°C. (C) To analyze receptor recycling, biotin-labeled and 3A6-treated A2774 cells were allowed to internalize ALCAM/CD166 for 40 minutes and were treated with GSH. Cells were then re-incubated at 37°C for 20 minutes to chase internalized receptors, and treated with (+) or without (–) the reducing agent to remove the label from molecules re-expressed to the cell surface. Cell lysis and I/F8-sepharose bead immunoprecipitation followed. Internalized ALCAM/CD166 recycles to the cell surface. (D) Kinetics of recycling. NHS-SS-biotin-labeled internalized receptors were chased by re-incubation at 37°C for different time points (t) before the second GSH treatment and immunoprecipitation. Immunoprecipitates were resolved by SDS-PAGE and revealed by western blot analysis with HRP-streptavidin and chemiluminescence.

View larger version (96K): [in a new window]   Fig. 8. Internalization kinetics of the I/F8/RIP immunotoxin. A2774 cells were incubated with either I/F8/RIP (large and small left-hand panels) or RIP alone (small right-hand panels) at 4°C in the presence of the anti-RIP mAb CY12.14 as dimerizing agent. Cells were then left on ice (A,B) or transferred to 37°C for different times to induce internalization (C,D,E), and were subsequently untreated (small panels) or acid-washed to strip the surface-bound molecules (large panels). Samples were then fixed, permeabilized, stained by FITC-labeled secondary antibody and nuclei were counterstained with Propidium Iodide. Samples were visualized by using an Olympus confocal laser microscope system. Crosslinking the immunotoxin with anti-RIP mAb allowed the visualization of the internalized fraction at concentrations as low as 1 x10–8 M I/F8/RIP (about 0.2 µg/ml). This amount of immunotoxin was used, as it is the highest concentration that still shows a 100-fold difference in toxicity when compared to free RIP. Original magnification, 600 x.

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