doi: 10.1242/10.1242/jcs.00118
Lipid rafts and the local density of ErbB proteins influence the biological role of homo- and heteroassociations of ErbB2
Peter Nagy1,2,3,
György Vereb1,
Zsolt Sebestyén1,
Gábor Horváth1,
Stephen J. Lockett4,*,
Sándor Damjanovich1,2,
John W. Park5,
Thomas M. Jovin3 and
János Szöll
si1,
1 Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, POB 39, Debrecen H-4012, Hungary
2 Cell-biophysical Workgroup of the Hungarian Academy of Sciences, University of Debrecen, POB 39, Debrecen H-4012, Hungary
3 Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen D-37077, Germany
4 Bioimaging Group, Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA
5 Department of Medicine, Division of Hematology/Oncology, University of California at San Francisco, San Francisco, CA 94143, USA
* Present address: NCI-FCRDC, Boyle Street, Frederick, MD 21702, USA
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Fig. 2. Membrane areas with anomalously high ErbB2 homoassociation colocalize with membrane domains with high ErbB2 and comparatively low ErbB3 densities. SKBR-3 breast tumor cells were labeled with FITC-4D5 and Cy3-4D5 against ErbB2, and with unlabeled H3.90.6 anti-ErbB3 antibody followed by secondary labeling with a Cy5-tagged Fab. Images A and B show the fluorescence intensity distribution of Cy3-4D5 (ErbB2) and the Cy5-tagged secondary Fab (ErbB3), respectively. The FRET efficiency for ErbB2 homoassociation is displayed in image C. The areas inside the white polygons in image C have the highest FRET values. The same areas are marked with red polygons in images A, B, D-F. These areas have very high ErbB2 density and comparatively low ErbB3 density. The areas at the head of the arrows have the highest ErbB2 and ErbB3 densities, but ErbB2 homoassociation in these pixels is lower than inside the marked areas. Image A was thresholded, and pixels with high ErbB2 fluorescence intensity are white in image D, whereas pixels with intensity below the threshold are black. Image B was `bithresholded': two intensity values were determined, and only pixels whose intensity is between the two threshold values are white in image E, all other pixels are displayed in black. Threshold values were adjusted so that white pixels in image E have low ErbB3 intensities, but still above a certain level. Pixels with intensity values lower than the lower threshold are background pixels, and they have to be excluded from the analysis. In image F a pixel is displayed in white if the pixel is white in both image D and E. The distributions of white areas in image F correlate with pixels with high ErbB2 homoassociation in image C. Bar, 1 µm.
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Fig. 1. Dependence of ErbB2 homoassociation on local parameters. SKBR-3 breast tumor cells were labeled with FITC-4D5 and Cy3-4D5 against ErbB2 to measure the homoassociation of ErbB2; ErbB3 was labeled with Cy5-tagged secondary Fab following labeling with H3.90.6. The homoassociation of ErbB2 was measured with the donor pbFRET method, and it was correlated with the local density of ErbB2 and ErbB3 on a pixel-by-pixel basis. The average FRET efficiency for ErbB2 homoassociation is plotted as a function of the local density of ErbB2 and ErbB3.
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Fig. 3. The relationship between lipid rafts and the small- and large-scale clustering of ErbB2. (A,B) SKBR-3 cells were labeled with FITC-CTX-B, Cy3-4D5 and Cy5-4D5 Fab. The FITC (green) and Cy3 (red) channels are overlaid in image A. Yellow areas correspond to colocalization between CTX-B-labeled lipid rafts and ErbB2 clusters. The homoassociation of ErbB2 was calculated using the acceptor photobleaching FRET method, and the FRET efficiency is displayed in panel B. FRET efficiency is color-coded according to the color-scale. The white boxes mark areas with inverse correlation between the local CTX-labeling density and ErbB2 homoassociation: the greener the area is in image A, the lower the FRET efficiency is in the corresponding part of the cell in image B. (C) Heregulin-stimulated SKBR-3 cells were labeled with FITC-CTX-B and Cy3-4D5 Fab. (D) SKBR-3 cells were treated with FITC-CTX-B at 37°C for 30 minutes, and they were subsequently labeled with Cy3-4D5 Fab. (E) SKBR-3 cells were pretreated with FITC-CTX-B and then incubated with Cy3-4D5 antibody at 37°C for 30 minutes. In part A, C and E the FITC image (green) is overlaid on the Cy3 (red) image. Yellow areas correspond to significant colocalization between lipid rafts and ErbB2 clusters. Bar, 1 µm.
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Fig. 4. Lipid rafts influence the homoassociation of ErbB2. Lipid rafts were visualized with FITC-CTX-B, and ErbB2 was labeled with Cy3-4D5 and Cy5-4D5 antibodies in order to measure its homoassociation with the acceptor photobleaching FRET method. FRET efficiency was averaged in pixels with the same FITC-CTX fluorescence intensity, and the average FRET efficiency is plotted as a function of lipid raft labeling.
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Fig. 5. Relationship between caveolin, GM1-enriched domains and ErbB2 clusters. (A) Quiescent SKBR-3 cells were labeled with FITC-CTX-B (green channel) and with anti-caveolin antibody sc-894 followed by secondary staining with Cy3-conjugated goat anti-rabbit immunoglobulin to visualize sc-894 labeling (red channel). (B) Quiescent SKBR-3 cells were stained against ErbB2 (with Alexa488-4D5, green channel) and against caveolin (as in part A, red channel). (C) SKBR-3 cells were treated with FITC-CTX-B for 30 minutes at 37°C (green channel), and then stained against ErbB2 (Cy5-4D5, blue channel) and against caveolin (as in part A, red channel). Bar, 1 µm.
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Fig. 6. CTX-induced effects in the association properties and biological functions of ErbB2. (A) The homoassociation of ErbB2, and its heteroassociation with ErbB1 and ErbB3 was measured with flow cytometry on control cells (black bars) and cells pretreated with 8 µg/ml CTX-B at 37°C for 30 minutes (white bars). Error bars indicate the standard error of the mean calculated from three independent experiments (*P<0.01). (B) SKBR-3 cells were seeded in 6-well plates and starved for 48 hours. CTX pretreatment was carried out in the presence of 8 µg/ml CTX-B at 37°C for 30 minutes, and then cells were stimulated with EGF or heregulin for 10 minutes. The tyrosine phosphorylation of ErbB2 (left graph) and Shc (right graph) was measured in control (black bars), EGF-stimulated (white bars) and heregulin-stimulated (right hatched bars) samples. Membranes labeled with anti-phosphotyrosine antibody were stripped and reprobed with anti-ErbB2 or anti-Shc antibody. The phosphotyrosine content of the bands was normalized to the amount of protein (ErbB2 or Shc) immunoprecipitated and to the control samples. Asterisks indicate statistical significance of Student's t-test performed on non-stimulated and growth factor-stimulated samples (*P<0.1, **P<0.05). Error bars indicate the standard error of the mean calculated from three independent measurements. (C) Control ( ) and CTX-pretreated ( ) cells were incubated with unlabeled 4D5 antibody for 40 minutes. Samples were taken every 10 minutes, and cell surface ErbB2 was labeled with Alexa488-7C2. The fluorescence intensity of Alexa488-7C2-labeled cells was measured with flow cytometry. Error bars indicate the standard error of the mean (n=3). (D) Control and CTX-pretreated SKBR-3 cells were incubated with Alexa488-4D5 antibody at 37°C for 30 minutes. Cells were imaged using confocal microscopy, and vertical slices of the cells are shown. The approximate position of the cell membrane is marked with a black dashed line (bar, 1 µm). (E) 50,000 cells were seeded in culture dishes. Control SKBR-3 cells and cells treated with 4D5, CTX-B and both with 4D5 and CTX-B were cultured for 5 days. Treatment with the antibody and CTX-B was carried out as described in Materials and Methods. At day 5 the cell surface expression of ErbB2 was measured with flow cytometry by labeling cells with Alexa488-7C2. The black bars show the mean fluorescence intensity after subtraction of autofluorescence. The number of cells is shown by the white bars. Error bars indicate the standard error of the mean (*P<0.05 compared with the corresponding control, n=3).
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Fig. 7. Model for the association of ErbB proteins inside and outside clusters. The extent of ErbB2-ErbB2 and ErbB2-ErbB3 associations depend on the relative expression levels of the proteins: high local ErbB3 density decreases ErbB2 homoassociation. Lipid rafts, which were identified as GM1-enriched domains labeled by CTX-B in our studies, colocalize with ErbB protein clusters. Although the high local concentration of ErbB proteins inside clusters favors their spontaneous activation (e.g. the formation of highly active ErbB2 homodimers), lipid rafts keep the activation of ErbB2 under control by limiting the homodimerization of ErbB2. By contrast, ErbB proteins are maintained in a signaling competent form inside rafts (i.e. heregulin-responsive ErbB2-ErbB3 heterodimers are present), which are disassembled if ErbB proteins are removed from them. CTX-B treatment induces migration of GM1-enriched domains into caveolae and disrupts ErbB2-ErbB3, but not ErbB2-ErbB2 or ErbB2-ErbB1 dimers.
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© The Company of Biologists Ltd 2002
