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doi: 10.1242/10.1242/jcs.00036

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Biosynthetic FGF-2 is targeted to non-lipid raft microdomains following translocation to the extracellular surface of CHO cells

André Engling^*,1, Rafael Backhaus^*,1, Carolin Stegmayer¹, Christoph Zehe¹, Claudia Seelenmeyer¹, Angelika Kehlenbach², Blanche Schwappach², Sabine Wegehingel¹ and Walter Nickel^1,

¹ Biochemie-Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany
² Zentrum für Molekulare Biologie Heidelberg (ZMBH), Im Neuenheimer Feld 282, 69120 Heidelberg, Germany

View larger version (83K): [in a new window]   Fig. 1. Characterization of the cell lines CHOFGF-2-GFP, CHOGFP-FGF-2 and CHOGFP. The model cell lines generated to study non-conventional export of FGF-2 were characterized with regard to genomic cDNA integration (A), western blot analysis of doxicyclin-dependent protein expression (B), analysis of doxicyclin-dependent protein expression based on fluorescence microscopy (C-H), analysis of doxicyclin-dependent protein expression based on FACS (I-K). (A) PCR analysis: CHOFGF-2-GFP (lanes 1,4); CHOGFP-FGF-2 (lanes 2,5); CHOGFP (lanes 3,6). Lanes 1-3 represent PCR reactions using genomic DNA as template isolated from the cell lines indicated; lanes 4-6 represent PCR reactions using the original retroviral plasmids as template (positive controls). (B) Western blot analysis: CHOFGF-2-GFP (lanes 1,2); CHOGFP-FGF-2 (lanes 3,4); CHOGFP (lanes 5,6). Total cell lysates (20 µg protein/lane) were subjected to SDS-PAGE followed by a western blot analysis using affinity-purified anti-GFP antibodies. Lanes 1, 3 and 5 correspond to cell cultures incubated in the absence of doxicyclin; lanes 2, 4 and 6 correspond to cultures incubated in the presence of doxicyclin. (C-H) Fluorescence microscopy analysis: CHOFGF-2-GFP (C,F); CHOGFP-FGF-2 (D,G); CHOGFP (E,H). Panels C, D and E represent cell cultures incubated in the absence of doxicyclin; panels F, G and H represent cell cultures incubated in the presence of doxicyclin. (I-K) FACS analysis: CHOFGF-2-GFP (I); CHOGFP-FGF-2 (J); CHOGFP (K). The cell populations grown in the absence of doxicyclin are shown in white, those grown in the presence of doxicyclin are shown in grey.

View larger version (43K): [in a new window]   Fig. 2. Biochemical analysis of FGF-2 fusion protein secretion. The various cell lines indicated were analyzed biochemically with regard to secretion of the reporter molecules (A). Cells were grown in the presence of doxicyclin and heparin for 48 hours at 37°C. FGF-2-GFP and GFP-FGF-2 were affinity-purified from detergent cell extracts and the medium by using heparin sepharose. 1% (cells) and 15% (medium) of the eluates were subjected to SDS-PAGE. In case of CHOGFP cells, 1% of both cells and medium were directly subjected to SDS-PAGE (the amount of the medium loaded onto the gel had to be reduced to 1% of the total material because of the high protein concentration). Affinity-purified anti-GFP antibodies were used to detect the reporter molecules. Even after prolonged exposition, no GFP signal could be observed in lane 6. To analyze whether FGF-2-GFP is released by a specific mechanism, CHOFGF-2-GFP cells were grown for 48 hours at 37°C in the presence of doxicyclin, 125 µg/ml heparin and 25 µM ouabain, a drug known to inhibit FGF-2 export (B). The samples were processed as described above.

View larger version (30K): [in a new window]   Fig. 3. A novel experimental system to quantitatively determine FGF-2 export from living CHO cells. CHOFGF-2-GFP cells were grown for 18 hours at 37°C under the conditions indicated followed by dissociation from the culture plates by using a protease-free protocol. The cell suspension was then processed for the FACS analysis under the conditions indicated. Panels A-E represent dot blots where total GFP-derived fluorescence was blotted against cell surface-derived PE fluorescence. (A) Cells grown in the absence of doxicyclin. (B) Cells grown in the presence of doxicyclin and processed with anti-GFP antibodies. (C) Cells grown in the presence of doxicyclin and processed with anti-GFP antibodies. (D) Cells grown in the presence of doxicyclin followed by a wash procedure using a heparin-containing buffer and antibody processing. (E) Cells grown in the presence of doxicyclin followed by trypsin digestion and antibody processing. Panels F and G represent the corresponding histograms of GFP-derived fluorescence and PE-derived cells surface fluorescence, respectively. The colours correspond to the conditions shown in panels A-E.

View larger version (12K): [in a new window]   Fig. 4. Translocation to the cell surface of FGF-2 fusion proteins depends on the FGF-2 domain and is compatible with both N- and C-terminal GFP tagging. CHOFGF-2-GFP, CHOGFP-FGF-2 and CHOGFP cells were grown in the presence of doxicyclin for 18 hours at 37°C followed by FACS processing, including antibody treatment, as described in Materials and Methods. (A) Quantitative comparison of GFP-derived fluorescence. (B) Quantitative comparison of PE-derived cell surface fluorescence. For both GFP- and PE-derived fluorescence, the signal produced by CHOFGF-2-GFP cells was set to 100. The results shown are representative of two independent experiments.

View larger version (12K): [in a new window]   Fig. 5. Characterization of FGF-2-GFP export with regard to kinetics, unspecific release and inhibition by ouabain. (A) Kinetic analysis of FGF-2-GFP export. CHOFGF-2-GFP cells were grown in the presence of doxicyclin for the times indicated followed by FACS processing, including antibody treatment, as described in Materials and Methods. The raw data have been subjected to a weighted curve fit and are representative of two independent experiments. (B) CHOFGF-2-GFP cells were grown in the absence of doxicyclin followed by the addition of various amounts of a supernatant derived from homogenized CHOFGF-2-GFP cells that were grown for 48 hours in the presence of doxicyclin (lanes 1-5). Based on cell number, 0% (lane 1), 2.5% (lane 2), 5% (lane 3), 7.5% (lane 4) and 10% (lane 5) of this supernatant was added to CHOFGF-2-GFP cells grown in the absence of doxicyclin. The PE-derived FGF-2-GFP cell surface signal was then compared with the corresponding signal of CHOFGF-2-GFP cells grown for 48 hours in the presence of doxicyclin (set to 100%, lane 6). Lanes 7 and 8 refer to experiments under the same conditions as those in lane 6 with the exception that during the whole course of the experiment, 1 µM and 5 µM ouabain, respectively, were added to the culture medium. The data are representative of two independent experiments.

View larger version (24K): [in a new window]   Fig. 6. FGF-2 externalization is not limited by the availability of cell surface HSPGs. CHOFGF-2-GFP cells were grown for 18 hours at 37°C in the presence (coloured curves in A and B) or absence (white curves in A and B) of doxicyclin. At the end of the incubation, various amounts of recombinant His6-tagged FGF-2 were added to the culture medium (green, 0.1 µg/ml; pink, 0.25 µg/ml; blue, 0.5 µg/ml; orange, 1 µg/ml; dark blue, 2 µg/ml) followed by an incubation for 60 minutes at 37°C. The red curves in A and B represent a standard FGF-2 secretion experiment in the absence of exogenously added FGF-2. The cell suspension was processed for the FACS analysis as described in the legend to Fig. 3. In this experiment, affinity-purified anti-FGF-2 antibodies were used to detect both cell surface FGF-2 and FGF-2-GFP. (A) GFP-derived fluorescence. (B) FGF-2 cell surface staining.

View larger version (71K): [in a new window]   Fig. 7. Translocation of FGF-2-GFP to the extracellular surface of the plasma membrane, as determined by confocal microscopy. CHOFGF-2-GFP and CHOGFP cells were grown on glass coverslips for 24 hours at 37°C in the absence or presence of doxicyclin. Where indicated, cells were washed with PBS containing 125 µg/ml heparin. Following fixation using paraformaldehyde, cells were processed with affinity-purified anti-GFP antibodies and secondary antibodies coupled to an Alexa546 fluorophore. The specimens were embedded using Fluoromount and viewed with a Zeiss LSM 510 confocal microscope. The results shown are representative of four independent experiments.

View larger version (108K): [in a new window]   Fig. 8. Identification of FGF-2-GFP-positive microdomains on the extracellular surface of CHO cells. CHOFGF-2-GFP cells were grown on glass coverslips for 24 hours at 37°C in the presence of doxicyclin. Processing for confocal microscopy was performed as described in the legend of Fig. 6. (A) Merged image of 16 confocal planes spanning the whole depth of the cells. (B) A confocal plane close to the bottom of the cells where they are attached to the glass coverslips.

View larger version (48K): [in a new window]   Fig. 9. FGF-2-GFP-positive microdomains are distinct from lipid rafts. CHOFGF-2-GFP cells were grown on large culture plates for 48 hours at 37°C in the presence of doxicyclin. Following a wash procedure using PBS the cells were scraped off the culture plates in a sucrose-containing buffer. Cell breakage was achieved by using a balch homogenizer followed by differential centrifugation at 1000 g and 5000 g to sediment nuclei and cell debris. The resulting supernatant was loaded on top of a 20% (w/v) sucrose cushion and centrifuged for 60 minutes at 100,000 g in order to collect microsomal membranes freed of cytosolic proteins. The membrane sediment was resuspended in PEN buffer containing 1% (w/v) Triton X-100 at 4°C. While being resuspended several times using a 100 µl tip, the membrane suspension was kept on ice for 30 minutes. The samples were then divided and either subjected to ultracentrifugation in order to sediment detergent-insoluble complexes or adjusted to 40% (w/v) sucrose followed by flotation in a linear sucrose gradient. (A) Detergent-soluble fraction (lane 1), detergent-insoluble fraction (lane 2). (B) 14 fractions of the linear flotation gradient (lanes 1-14) with lane 1 containing the most dense sucrose fraction and lane 14 containing the lightest fraction. In the case of FGF-2-GFP and p23 detection, 60% of each fraction was TCA-precipitated and applied to the gel; in the case of caveolin-1, 15% of each fraction was TCA-precipitated and applied to the gel.

View larger version (9K): [in a new window]   Fig. 10. Both endogenous and exogenously added FGF-2 do not stimulate proliferation activity of CHO cells. CHOFGF-2-GFP cells were spread on culture plates at a confluency of about 5-10%. Cell proliferation was monitored for 48 hours in the presence of recombinant His6-FGF-2 (5 µg/ml; dotted line); in the presence of doxicyclin to induce FGF-2-GFP expression and externalization (solid line); or in the absence of doxicyclin as a control condition (dashed and dotted line).

View larger version (63K): [in a new window]   Fig. 11. Intercellular spreading of secreted FGF-2-GFP. CHOFGF-2-GFP and CHOMCAT-TAM2 cells were cultured on glass coverslips in a 1:1 ratio. Following incubation for 24 hours at 37°C in the presence of doxicyclin, the cells were fixed with PFA and processed with affinity-purified anti-GFP antibodies. Primary antibodies were detected with anti-rabbit IgG antibodies coupled to Alexa546. The specimens were viewed using a Zeiss LSM 510 confocal microscope.