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First published online 15 July 2008
doi: 10.1242/jcs.028753

Journal of Cell Science 121, 2519-2528 (2008)
Published by The Company of Biologists 2008
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EGF induces coalescence of different lipid rafts

Erik G. Hofman1, Mika O. Ruonala2, Arjen N. Bader3, Dave van den Heuvel3, Jarno Voortman1, Rob C. Roovers1, Arie J. Verkleij1, Hans C. Gerritsen3 and Paul M. P. van Bergen en Henegouwen1,*

1 Department of Cellular Architecture and Dynamics, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands
2 Center for Membrane Proteomics, University of Frankfurt, Biocenter, Frankfurt am Main, Germany
3 Department of Molecular Biophysics, Utrecht University, Utrecht, The Netherlands


Figure 1
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  		Fig. 1. Characterization of anti-EGFR nanobodies. (A) Immunoprecipitation of EGFR with nanobodies. HER14 cell lysates were prepared as described in Material and Methods and incubated for 1 hour at 4°C with Talon beads preloaded with the indicated anti-EGFR nanobodies, or with an non-specific nanobody (anti-GST). Bound proteins were separated by SDS-PAGE, analyzed by western blotting. Control lane (Cell lysate) is loaded with 10% of the lysate. (B) Selected nanobodies are non-agonistic. An equal number of serum-depleted HER14 cells was treated with either 8 nM EGF, 1 µM of the indicated nanobodies for 10 minutes, or mock-treated (no EGF), and immunoprecipitated EGFR was analyzed by western blotting. Activation of EGFR was determined with an antibody against phosphorylated tyrosine at position 1068 (pEGFR). Loading control was performed with anti-EGFR (EGFR). (C) Selected nanobodies bind specifically to EGFR. NIH 3T3 clone 2.2 cells, lacking detectable expression of EGFR, or HER14 cells were labeled at 4°C for 60 minutes with 100 nM EGb4-A594. In parallel, the cells were labeled with 8 nM EGF-A488 and nuclei were visualized with 4'-6-diamidino-2-phenylindole (DAPI; blue). (D) Different antagonistic activities of anti-EGFR nanobodies. HER14 cells grown in 96-well plates, were pre-incubated with biotinylated EGF, followed by increasing concentrations of indicated nanobodies or EGF. Binding of EGF-biotin on the cell surface was quantified using peroxidase-conjugated streptavidin, followed by addition of the substrate OPD.

 

Figure 2
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  		Fig. 2. Nanoscale colocalization of EGFR with GM1 gangliosides. (A-C) HER14 cells grown on coverslips were incubated on ice with 100 nM (A) of the donor probes anti-EGFR nanobody EGa1 or (B) EGb4 directly conjugated to Alexa-Fluor-488, or with (C) 20 µg/ml transferrin-A488 (Tf-A488) in the absence or presence of 1 µg/ml of the acceptor probe CTB-A594. (D) HER14 cells expressing GPI-GFP were incubated or not for 1 hour on ice with 100 nM EGb4-A594 (acceptor). After fixation with 4% formaldehyde, average lifetime values of GFP were determined as described in Materials and Methods. Left panels represent the distributions of the donor probes with or without acceptor probe. The lifetimes are shown in the middle panels in false colors. Mean fluorescent lifetime values ± s.e.m. (***P<0.0001) are presented in histograms on the right.

 

Figure 3
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  		Fig. 3. Cholesterol-dependence of the colocalization of GPI-GFP and EGFR with GM1. (A) Cholesterol-dependent colocalization of GPI-GFP with GM1. HER14 cells expressing GPI-GFP (donor) were incubated or not with 5 µg/ml nystatin for 30 minutes at 37°C and labeled or not with CTB-A594 (donor probe). The fluorescent intensity of GPI-GFP or CTB-A594 is shown in the left or middle panel, respectively. Fluorescent lifetime of GPI-GFP was analyzed as described in Materials and Methods and presented as false-colored images in the right panel. Histograms represent the mean lifetime values ± s.e.m. (***P<0.0001) of GPI-GFP determined under the indicated conditions. (B,C) Cholesterol-independent colocalization EGFR with GM1 gangliosides. HER14 cells were treated with (B) 5 µg/ml nystatin or (C) 2.5 µg/ml filipin for 30 minutes at 37°C. Cells were labeled with anti-EGFR nanobody EGa1-A488 and labeled or not with the acceptor probe CTB-A594. Lifetime values were determined as indicated in Materials and Methods, and mean lifetime values of ± s.e.m. (**P<0.001) are presented in histograms.

 

Figure 4
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  		Fig. 4. EGF does not affect colocalization of EGFR with GM1 gangliosides (A) EGFR is activated at 4°C. HER14 cells were stimulated with 8 nM EGF for indicated times and temperatures. Cell lysates were prepared and analyzed by western blotting for the presence of phosphorylated tyrosine at position 1068 (pEGFR). Actin staining was used as loading control. (B) Colocalization of activated EGFR with GM1. HER14 cells were incubated on ice with 8 nM EGF-488 (donor) in the absence or presence of 1 µg/ml acceptor probe CTB-A594. Confocal images representing the distribution of EGF-A488 are shown in the left panel. The lifetime of EGF-A488 was determined as described in Materials and Methods and presented in false colors in the middle panel. Mean lifetime values ± s.e.m. (***P<0.0001) were determined and are presented in the histogram on the right. (C) EGF does not alter colocalization of EGFR and GM1. HER14 cells were incubated with 100 nM EGb4-A488 and 1 µg/ml CTB-A594 for 1 hour on ice. Cells were then either treated with 8 nM EGF for 10 minutes or left untreated. After fixation and embedding, lifetime values of EGb4-A488 were analyzed as described in Materials and Methods and are presented as mean lifetime values ± s.e.m. (**P<0.001, ***P<0.0001) in the histogram on the right.

 

Figure 5
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  		Fig. 5. EGF-induced colocalization of EGFR and GPI-GFP. (A) Colocalization active EGFR with GPI-GFP. HER14 cells expressing GPI-GFP were incubated for 1 hour on ice with 8 nM EGF-Rhodamine (EGF-Rho) for 60 minutes, or mock medium (without EGF-Rho). After fixation and embedding, the average lifetime values ± s.e.m. (***P<0.0001) of GPI-GFP were determined as described in Materials and Methods and are presented in the histogram on the right. (B) HER14 cells expressing GPI-GFP were incubated for 1 hour on ice with 100 nM EGc5-A594. Activation with 8 nM EGF was performed on ice for 10 minutes. After fixation and embedding, mean lifetime values ± s.e.m. (***P<0.0001) of GPI-GFP were determined as described in Materials and Methods and are presented in the histogram. (C) HER14 cells expressing GPI-GFP were pre-incubated or not with 5 µg/ml nystatin for 30 minutes at 37°C, for 1 hour on ice in the absence or presence of 100 nM CTB-A594, and stimulated or not for 10 minutes with 8 nM EGF. After fixation and embedding, average lifetime values ± s.e.m. (***P<0.0001) of GPI-GFP were determined as described in Materials and Methods and are presented in the histogram. (D) HER14 cells expressing GPI-GFP were incubated for 1 hour on ice in the absence or presence of 100 nM CTB-A594 or CTB-biotin as control. Activation with 8 nM EGF was performed on ice for 10 minutes. After fixation and embedding, mean lifetime values ± s.e.m. (**P<0.001) of GPI-GFP were determined as described in Materials and Methods and are presented in the histogram. (E) EGF induced the recruitment of GPI-GFP into the detergent-free lipid raft fraction. HER14 cells expressing GPI-GFP were stimulated or not with 8 nM EGF for 10 minutes, and detergent-free lipid raft fractions were isolated as described in Materials and Methods. Presence of EGFR, GPI-GFP and MAP kinase was analyzed by western blotting, presence of GM1 was determined using a dot-spot assay with CTB-HRP.

 

Figure 6
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  		Fig. 6. Model showing the coalescence of different lipid rafts. In resting cells, GM1 forms a lipid shell that surrounds the EGFR by binding to the ectodomain, and colocalizes in a cholesterol-dependent manner with GPI-GFP. Stimulation of the cell with EGF results in the coalescence of these two different microdomains possibly leading to the formation of signaling platforms and the initiation of EGFR internalization.

 

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