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Heterogeneity of signal transduction at the subcellular level: microsphere-based focal EGF receptor activation and stimulation of Shc translocation

Roland Brock^* and Thomas M. Jovin

Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany
^* Present address: Institute of Organic Chemistry, University of Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany (e-mail: roland.brock{at}uni-tuebingen.de)

View larger version (89K): [in a new window]   Fig. 1. Internalization of EGF-coated microspheres. Shown are stereo-images of GFP fluorescence of E-CHO cells (expressing the EGFR-GFP fusion protein) after 2 minutes (A), 5 minutes (B), 8 minutes (C) and 20 minutes (D) incubation with the microspheres. By 2 minutes most microspheres were completely engulfed by the plasma membrane. At 8 minutes, the distribution of fluorescence around the microspheres appeared less fuzzy. At 20 minutes some microspheres had translocated to the basal side of the cell. The stereo-images were processed in such a way that the cell-coverslip contact is oriented towards the observer. The microspheres themselves were not fluorescent, but could be visualized in the confocal reflection mode. The fluorescence stacks were deconvoluted as described in Materials and Methods.

View larger version (86K): [in a new window]   Fig. 2. Disposition of the plasma membrane about the microspheres after 2 minutes (A) and 8 minutes (B) incubation of E-CHO cells with EGF-coated microspheres. Each panel presents one confocal slice of the total image (left) and two sets of enlargements of the microspheres located in the regions-of-interest defined in the overviews. One xy-slice out of a total of 20, and xz- and yz-projections along the indicated lines are shown. Outlines of the morphology of the plasma membrane as derived from the fluorescence images are included. (A) Microspheres, located in an invagination of the plasma membrane reaching down nearly the entire thickness of the cell. In other cases, engulfment was complete at 2 minutes. (B) Microsphere internalized into the cell, concluded from the presence of fluorescence of the outer membrane above the microsphere. Deconvolution was carried out as described in Materials and Methods.

View larger version (73K): [in a new window]   Fig. 3. Inhibition of internalization of EGF-coated microspheres in the presence of the EGFR-specific tyrosine kinase inhibitor PD153035. (A) Stereo-images of GFP fluorescence of E-CHO cells 5 minutes after contact with EGF-coated microspheres. In contrast to Fig. 1, the apical face of the cells is oriented towards the observer. (B) Examples of plasma membrane morphology of E-CHO cells in contact with EGF-coated microspheres after 5 minutes incubation with the microspheres. The locations of the enlarged regions of interest 1-3 are boxed in the overview, showing one confocal slice. A set of three orthogonal sections is given in each case; the positions of the sections are indicated. The sampling frequency along the optical axis was 0.3 µm. The fluorescence stacks were deconvoluted as described in Materials and Methods.

View larger version (56K): [in a new window]   Fig. 4. Immunofluorescence microscopy with EGF-coated microspheres. (A) Sequestration of the EGF receptor. The dynamic range of the look-up-table was adjusted according to the fluorescence intensities in the cells. Thus, the bead-associated fluorescence appears to be saturated. (B,C) Double labeling of cells to probe for activation of the EGF receptor (B) by a monoclonal antibody specific for the activated isoform of the EGF receptor and (C) translocation of Shc to the activated EGF receptor. One slice out of a stack of eight is shown in each case, accounting for the different intensities of the signals at microspheres located in different focal planes. As in A, the dynamic range of the look-up-table was adjusted to display the fluorescence in the cells. Bar, 10 µm. The locations of the microspheres detected by confocal reflection are superimposed by white outlines.

View larger version (60K): [in a new window]   Fig. 5. Image processing protocols for isolation of the signals for EGFR activation and Shc translocation and generation of three-dimensional data sets. Schematics illustrating the immunofluorescence staining are included in the leftmost panels. The image processing protocol for the analysis of receptor activation only retains the signals colocalizing with the microspheres (colored pixels in the top center panel). The positions of the microspheres are outlined in red on one slice of the three-dimensional stack of confocal images. For Shc the signal at the microspheres as well as the Shc signal in the local environment are retained. The microspheres are again designated by red pixels. To illustrate the heterogeneous subcellular distribution of Shc more clearly an unsegmented enlargement is included in the bottom center panel. The colored ring around the microsphere-associated signal represents the local environment. An xz-projection through the image stacks is included for the enlarged areas. The final three-dimensional data set consists of (1) the level of EGFR activation, (2) the Shc signal in the local environment of a microsphere, and (3) the Shc signal colocalizing with the microsphere. The pseudocolor look-up table was chosen to better illustrate which data were isolated by the image processing procedure.

View larger version (36K): [in a new window]   Fig. 6. Time dependence of (A) EGFR activation and (B) Shc translocation. Frequency histograms after 2, 5, 8 and 20 minute incubations of A431 cells with EGF-coated microspheres. Insets show mean receptor activation as well as Shc translocation expressed in arbitrary fluorescence units (a.u.). Values are means ± 1 s.d. of three independent experiments.

View larger version (24K): [in a new window]   Fig. 7. Time-dependent correlation of Shc translocation with the activation of EGFR. The data sets from three different experiments were subdivided for low, medium and high levels of Shc in the local environment. The intervals for the subsets were adjusted to roughly equal numbers of events per subset and were the same for all experiments. Values within each subset were smoothed by a moving average with a window size of five to further compensate for the effect of the local Shc concentration on the analysis of receptor-dependent Shc translocation. The arrow indicates those data points lying outside the major population that were caused by a high Shc concentration in the local environment. The values for EGFR activation do not extend below 20 a.u. because of thresholding in the image processing procedure.