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)
Author for correspondence (e-mail: tjovin{at}mpc186.mpibpc.gwdg.de)
Accepted April 2, 2001
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SUMMARY |
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Key words: Tyrosine kinase inhibitor PD153035, Confocal laser scanning microscopy, Immunofluorescence, Digital image processing, QMRA
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Introduction |
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The EGFR is prototypic for the family of receptor tyrosine kinases (RTK; Ullrich and Schlessinger, 1990), with protein-protein interactions constituting the basis for the transduction of intracellular signals (Alroy and Yarden, 1997; Olayioye et al., 2000). The adaptor protein Shc (Pelicci et al., 1992) plays a central role in signaling via the EGFR, binding the activated receptor through its N-terminal phosphotyrosine binding domain (PTB) and C-terminal SH2 domain (SH2) with subsequent phosphorylation of tyrosine residues in the central collagen-homology domain 1 (CH1; Gotoh et al., 1997). These serve in turn as recognition sites for additional downstream components, among them the adaptor protein Grb2 (Egan et al., 1993), finally leading to activation of p21ras and signaling through the cascade of mitogen-activated kinases (MAPK; Blumer and Johnson, 1994). Analyses of the distribution of Shc in unstimulated cells by confocal immunofluorescence microscopy and electron microscopy (Lotti et al., 1996) have revealed a perinuclear and reticular distribution, reflecting an association with the cytoplasmic side of the rough endoplasmic reticulum, the nature of which is unknown. Shc is therefore a candidate protein whose signaling potential may vary at the subcellular level. Since Shc binds to the activated EGFR directly but also participates in other signaling events, there is a conceptual basis for both correlation of translocation to the activation state of the EGFR and modulation of this activity by downstream activities.
Microspheres with tightly coupled ligands constitute highly localized, non-diffusive stimuli for the cell surface receptor and have been employed in a number of cellular systems. For example, bFGF-coated microspheres led to clustering of acetylcholine-receptors in fibroblasts (Peng et al., 1991; Baker et al., 1992). Sequestration of receptor tyrosine kinases to sites of integrin signaling (Miyamoto et al., 1996) as well as long-range effects of integrin- and cadherin-coated microspheres on cellular phosphotyrosine levels (Levenberg et al., 1998) have also been demonstrated. Image analysis in these studies was mostly semi-quantitative, discriminating in a binary fashion between microspheres positive or negative for the given protein (Miyamoto et al., 1996). Levenberg and colleagues (Levenberg et al., 1998) introduced a quantitative form of image analysis, in which the investigator manually selects regions-of-interest in subregions of cells. In other studies, colocalization was quantitated by disruption of the beads from the cells and subsequent SDS-polyacrylamide gel electrophoresis (Plopper and Ingber, 1993; Miyamoto et al., 1995). As the beads were 5-12 µm in diameter, it is conceivable that they may have led to a significant perturbation of the surface and internal structure of the cell. Such beads are also too large for correlating protein colocalizations with a spatial resolution adequate for assessing subcellular heterogeneity.
In the quantitative microsphere recruitment assay (QMRA) described and used in this report, a growth factor is covalently coupled to microspheres that are small (1 µm diameter) compared to the size of the cell. The cells are incubated with the beads for different durations of time. Activation of cell surface receptors and recruitment of downstream factors to the activated receptors are investigated by confocal immunofluorescence microscopy. A comprehensive quantitative analysis is achieved by image-processing protocols that filter the microsphere-associated signals from noise and background and automatically quantitate large numbers of objects. Since cellular integrity is fully preserved, a quantitative assessment of intracellular processes in the face of subcellular heterogeneity is possible. The specific and rapid internalization of ligand-coated microspheres within minutes, and the inhibition of these processes by receptor-specific tyrosine kinase inhibitors, indicate that such small microspheres elicit physiological responses that are normally observed with soluble ligands. The observation of some of these phenomena was greatly facilitated by employing cells expressing a fusion protein of EGFR with a Green Fluorescent Protein (GFP) (Brock et al., 1999a; Brock et al., 1999b).
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MATERIALS AND METHODS |
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Generation of EGFR-GFP fusion proteins and transfected cell lines
The EGFR-GFP fusion protein was generated as described elsewhere (Brock et al., 1999a). The EGFR was derived from an EGFR cDNA (Ullrich et al., 1984) in pcDNA3 (Invitrogen, Carlsbad, CA, USA; obtained from Y. Yarden) and cloned into the pEGFP-N3 plasmid (Clontech, Heidelberg, Germany). CHO cells were seeded in 35 mm Petri dishes at a confluency of 10-15%. The following day, the DMEM was replaced with 0.8 ml Optimem (Gibco Life Technologies, Eggenstein, Germany), supplemented with antibiotics. 1 µg of pEGFP-N3 plasmid DNA and 3 µl of a non-commercial transfection agent (H. Eibl, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany) or 6 µl Lipofectin (Gibco) were incubated separately with 100 µl of Optimem for 15 minutes, followed by another 30 minutes after mixing of the two solutions at room temperature. Transfection took place in a final volume of 1 ml over 24 hours. Cells expressing the fusion construct (E-CHO) were selected in medium supplemented with 0.4 mg/ml G418 (Gibco) and two rounds of cell sorting (Epics Elite, Coulter Electronics, Krefeld, Germany) at 2-week intervals. The structural and functional integrity of the EGFR-GFP fusion protein localized to the plasma membrane has been characterized in detail (Brock et al., 1999a; Brock et al., 1999b).
Preparation of liganded microspheres
Natural murine EGF was purchased from IC Chemikalien (Ismaning, Germany). Carboxy-functionalized super-paramagnetic 1 µm beads (SERA-MAG, Seradyn, Indianapolis, USA) were activated with 0.1 M sulfo-NHS (N-hydroxysulfosuccinimide; Pierce, Rockford, USA), 0.1 M EDC (1, ethyl(-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; Pierce) in 0.1 M MES (Sigma, Deisenhofen, Germany) buffer, pH 5, for 1 hour at room temperature. After washing twice in 0.1 M MES, the microspheres were equilibrated in coupling buffer (0.1 M sodium phosphate, pH 8). The coupling reaction was carried out at 4°C overnight with 50 µg EGF in 30 µl coupling buffer per 6 µl of the 5% bead slurry with constant agitation. Beads used as negative controls in immunofluorescence experiments were incubated with ethanolamine or BSA (Sigma). Finally, the beads were washed twice with coupling buffer and then thoroughly with PBS after quenching remaining activated groups with 1 M ethanolamine for 2 hours at room temperature. The microspheres were stored in PBS with 0.1% sodium azide. The presence of intact EGF on the surface of the microspheres was demonstrated in a bead agglutination assay (Dezelic et al., 1971) with an anti-murine EGF rabbit polyclonal antibody (Sigma). A nonspecific antibody was inactive.
Preparation of fluorescently labeled antibodies
Mouse monoclonal antibody against the activated isoform of the human EGFR (anti-EGFRa) and rabbit polyclonal anti-Shc antibody were purchased from Transduction Laboratories (Lexington, KY, USA). BSA present in the antibody preparations was removed prior to fluorescent labeling with pre-packed 1 ml protein-G columns (AGS, Heidelberg, Germany). The antibodies were concentrated to 100 µg/ml in Microcon 100 microconcentrators (Amicon, Witten, Germany) and labeled with a tenfold molar excess of the NHS-esters of the indocyanine dyes Cy3 and Cy5 (Amersham Buchler, Braunschweig, Germany) (Southwick et al., 1990) for 30 minutes at room temperature in 100 mM sodium bicine, pH 8 (Sigma). The reaction was quenched with 5 µl 1 M glycine. Unreacted dye was removed on an Econo-Pac 10DG column (BioRad, Richmond, CA, USA). The antibody fraction was concentrated in Microcon 100 microconcentrators and the molar labeling ratios (dye:antibody) were determined with UV spectroscopy according to the specifications of the supplier. They were in the ranges of 1-3 for different preparations. The antibodies were stored in 50% glycerol, 0.1% BSA, 0.1% sodium azide at -20°C. A single preparation of each of the antibodies was used for all of the experiments.
Microsphere experiments and immunofluorescence microscopy
Cells were seeded on 12 mm diameter glass coverslips. For measurements of receptor activation after 2 days of culture the cells were serum starved for 24 hours and used at a confluency of 70-90%. The cells were washed twice in HBS (Hepes-buffered saline: 135 mM NaCl, 10 mM KCl, 0.4 mM MgCl2, 1.0 mM CaCl2, 5.6 mM glucose, 0.1% BSA, 10 mM Hepes (Sigma), pH 7.4) and warmed to 37°C in a humid incubation chamber with 50 µl HBS for 5 minutes. To accelerate the sedimentation of the magnetic microspheres, magnets were placed underneath the coverslips in the humid incubation chambers. Microspheres were added in another 50 µl at a density of 1-10 per cell and the incubation was carried out over the indicated times. The cells were rinsed in ice-cold HBS and fixed in 3.7% paraformaldehyde for 5 minutes on ice, followed by 10 minutes at room temperature. The coverslips were washed three times with PBS, and once with 100 mM Tris-HCl, pH 7.4, 50 mM NaCl. The cells were permeabilized with 0.1% Triton X-100 (Fluka Sigma-Aldrich Chemie, Deisenhofen, Germany) in PBS for 10 minutes at room temperature, followed by three more washes with PBS. Incubations with fluorescently labeled primary antibodies were carried out at concentrations of approx. 1 µg/ml in 50 µl PBS/0.2% BSA for 1 hour at room temperature. The EGFR was probed with an anti-EGFR mouse monoclonal antibody (clone F4, Biomol Feinchemikalien, Hamburg, Germany) in combination with a Cy3-labeled goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Cells were washed four times with PBS for 15 minutes and mounted on microscope slides in 0.1 M Tris-HCl, pH 8.5, 25% (w/v) glycerol, and 10% Mowiol 4-88 (Hoechst Pharmaceuticals, Frankfurt, Germany) (Osborn and Weber, 1982). For high-resolution confocal analyses of the cellular morphology about the microspheres, E-CHO cells incubated with microspheres were fixed by incubation with PFA for 5 minutes at 4°C and 10 minutes at room temperature, followed by three 5 minute washes with HBS and a subsequent methanol fixation for 6 minutes at -20°C.
The EGFR-specific tyrosine kinase inhibitor PD153035 (Calbiochem-Novabiochem, Bad Soden, Germany) (Fry et al., 1994) was used at a concentration of 1 µM. Before addition of microspheres, cells were incubated in HBS supplemented with inhibitor, followed by PFA fixation in the presence of the inhibitor. A 5 mM DMF stock solution was stored at -20°C. Control microsphere experiments with DMF diluted 1:5000 were included.
Confocal laser scanning fluorescence microscopy was carried out with an LSM310 system (Carl Zeiss, Göttingen, Germany) and a 63x, 1.4 NA Plan-Apochromat oil immersion objective. GFP was excited at 488 nm with the internal Argon-ion laser and its emission selected through a 530DF30 bandpass filter (Omega Optical). For signaling experiments with A431 cells, Cy3 was excited with a 1 mW 543 nm He-Ne laser (PMS Electro-Optics, Boulder, CO, USA) and its emission selected through a 590DF35 bandpass filter (Omega Optics, Brattleborough, USA). Cy5 was excited with a tenfold attenuated 5 mW 633 nm He-Ne laser and detected through a LP665 long-pass filter (Carl Zeiss). The lateral and axial sampling frequencies varied between experiments and are given in the Results section. In most cases images were recorded with two- to fourfold oversampling in the x and y dimensions and oversampling to various degrees in the z dimension. The sampling time per frame was 2 seconds, with twofold frame-averaging for a stack of 20 slices and fourfold averaging for 10 slices. In all cases, reflection images were acquired in parallel with the fluorescence stacks for the identification of the microspheres, using the 100-fold attenuated 633 nm laser.
Image analysis
Image processing was carried out with SCIL-IMAGE (University of Amsterdam, TNO Institute of Applied Physics, Delft, The Netherlands). Before segmentation, deconvolutions employing Maximum a posteriori approaches based on Goods roughness (MAPPR) (Verveer and Jovin, 1998) and calculated point-spread-functions (PSF) (van de Voort and Brakenhoff, 1990) were carried out. The special functions for the quantitative analysis of bead-associated fluorescence were written in C and implemented in the SCIL-IMAGE package. The image analysis protocol performed a segmentation of the three-dimensional image stacks into bead-associated fluorescence, regions of fluorescence in the rest of the cell, and fluorescence in the local environment of the microspheres (in the case of Shc translocation). The local environment was selected as a region of closest vicinity to the microsphere that was deemed unaffected by changes in membrane morphology or protein recruitment. The image processing procedure is illustrated in Fig. 5 and a detailed description will be given elsewhere.
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RESULTS |
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Microsphere engulfment and internalization
The potential of the EGF-coated microspheres to internalize was a major concern with respect to downstream molecular events occurring after EGFR endocytosis (Vieira et al., 1996). Chemical modification via the N-terminal amino-group of murine EGF does not impair receptor binding or activation, as was demonstrated for fluorescently labeled EGF (Gadella and Jovin, 1995) or EGF coupled to solid supports (Ito et al., 1998). E-CHO cells were incubated with the EGF-coated microspheres for 2, 5, 8 and 20 minutes and analyzed with confocal microscopy followed by image deconvolution for axial resolution enhancement (Figs 1, 2) (Verveer and Jovin, 1998). After 2 minutes, most microspheres were already fully engulfed by the plasma membrane (Figs 1A, 2A). At 8 minutes internalization was complete (Figs 1C, 2B) and translocation towards the basal side of the cells was evident (Fig. 1D). To determine whether microsphere internalization required the activation of the receptors, E-CHO cells were incubated with microspheres in the presence of the EGFR-specific tyrosine kinase inhibitor PD153035 (Fry et al., 1994). In contrast to all other experiments, the engulfment of the beads was only partial (Fig. 3). In some cases, only receptor recruitment to the site of microsphere contact was apparent. The inhibitor was diluted 1:5000 from a DMF stock-solution. However, DMF alone had no effect on the engulfment process described above. Although not quantitated in detail, fewer microspheres were present in inhibitor-treated cells than in control samples.
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Time-dependence of signal transduction at the EGF-coated microspheres
The time course of receptor activation and Shc recruitment at the subcellular level was analyzed in more detail. Figs 6 and 7 summarize the data from three independent experiments, each scaled to compensate for quantitative differences due to instrument factors (laser intensity, pinhole adjustment) based on calibration samples.
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Control experiments analyzing the density of EGFR at the microspheres indicated that the kinetics of receptor activation and Shc translocation were unaffected by ongoing EGFR recruitment to the microspheres. Neither for A431 nor for E-CHO cells was a change of receptor density observed (not shown). Consistent with a qualitative assessment of the image data (Figs 1, 2) the receptors were already enriched at the microspheres after 2 minutes. No discernible depletion of receptors in the surrounding region was observed in either case, from which we conclude either that this effect was negligible or that long-range re-equilibration of receptors occurred very rapidly.
Correlation of EGF receptor activation, Shc translocation and the local Shc concentration at the subcellular level
For each microsphere a data set that included EGFR activation, local Shc concentration and Shc signal at the microspheres was extracted from the confocal images by the image processing protocol.
At 2 minutes the data points for EGFR activation clustered, without a perceivable correlation between EGFR activation and Shc translocation. However, a positive correlation was apparent at 5 and 8 minutes. By 20 minutes the positive correlation had vanished and the distribution of the data points widened. The values for receptor activation did not extend below a certain value, dictated by thresholding in the image processing procedure. Consistent with the assumption outlined above, high levels of local Shc concentration disguised the positive correlation of Shc translocation and receptor activation. To further compensate for the perturbation of the correlations by the local Shc concentration, the data within one data set were slightly smoothed by local averaging.
The scatter plots in Fig. 7 showed an increase of EGFR activation and Shc translocation with time as well as a build-up and an ultimate dissipation of a positive correlation between both parameters. In addition to shifting towards higher or lower values, the distributions of the 20 minute data broadened for all combinations of high and low receptor activation levels and Shc translocation. The subcellular build-up and dissipation of the correlation of Shc translocation with EGFR activation were characterized quantitatively by calculating the linear correlation coefficients between both values (Table 1). The qualitative results evident in the scatter plots were confirmed quantitatively. The correlation coefficients reached a maximum of 0.8 at 5-8 minutes and decreased to below the 2 minute value at 20 minutes. As expected, the correlation coefficients for the subpopulation with low local Shc concentrations were higher than for the other subpopulations.
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DISCUSSION |
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In the presence of the EGFR-specific tyrosine kinase inhibitor PD153035 the microspheres were only partially engulfed by the plasma membrane and no internalization occurred. Furthermore, the number of microspheres remaining on the cells after fixation and washing was lower than in untreated control samples.
We conclude that the 1 µm EGF-coated microspheres elicited a cellular response similar to that observed for soluble EGF, thus constituting a valid approach for studying internalization-dependent signaling mechanisms. It will be interesting to investigate in more detail the degree to which the endocytic machinery adapts to morphological structures that are much larger than a regular endocytic vesicle.
Time dependence of EGFR-dependent signal transduction in QMRA
The image processing protocol readily isolated the bead-associated fluorescence and the fluorescence in the local environment. The constancy of the mean values and CVs for EGFR-based fluorescence detected in A431 cells by immunofluorescence and in E-CHO cells by intrinsic GFP fluorescence provided a twofold quantitative validation of the image processing procedure, and of the microsphere-based approach for following the temporal course of activation and activation-dependent Shc recruitment.
At 2 minutes, considerable receptor activation was already present. Due to the experimental protocol, a zero time point could not be included. QMRA requires binding of the microspheres to the cells; receptor activation and Shc translocation are thus unavoidable. As receptor activation would also occur if cells were cooled to 4°C (Nesterov et al., 1990), it was impossible to define the starting point by a rapid temperature shift to 37°C. While one may argue that the very early molecular events are not accessible by QMRA, the molecular events of interest here, the build-up and dissipation of the correlations of EGFR activation and Shc translocation at the subcellular level, occurred between 2 and 20 minutes.
Receptor activation as well as Shc translocation persisted over the whole time course of the experiments. Compared to the 2 minute time point, mean receptor activation increased by a further 40%, in agreement with the relative 40% increase from 1 to 20 minutes reported previously using the same activation-specific antibody (Emlet et al., 1997). Prior reports based on EGF coupled to solid supports provided only a qualitative demonstration of EGFR activation (Ito et al., 1998). The mean Shc translocation increased by 75% from 2 to 8 minutes; no further increase was observed for the 20 minute time point. The increase in both the fractions of microspheres with the lowest and the highest signals is indicative of a heterogeneous pattern of deactivation and activation pathways proceeding in parallel by the latest time point.
Heterogeneous kinetics of EGFR-dependent signal transduction at the subcellular level
The microsphere-based subcellular analysis revealed a new level of signaling complexity at the subcellular level. The increase of EGFR activation and Shc translocation at the global level present in the frequency histograms was in contrast to a build-up and dissipation of a correlation for Shc translocation with EGFR activation at the subcellular level (Table 1) and by the widening of the distributions for both values (Table 2).
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The correlation between receptor activation and Shc translocation was strongest at 5 to 8 minutes and decreased by 20 minutes. This finding reveals a time-dependent decoupling of EGFR activation and Shc translocation and points to additional mechanisms that influence the kinetics of EGFR activation and Shc translocation at later time points. Because of the impact of the local Shc concentration on the Shc signal at the microsphere, the absolute values of the correlation coefficients will tend to underestimate the correlation of receptor activation and Shc. That is, the correlation coefficients for the subpopulation of microspheres with low Shc concentration in the local environment will represent more closely the direct molecular association of receptor activation and Shc recruitment. The relative width of the Shc distributions grew by only 25%, i.e. signaling coherence was preserved. The observation that the CV for Shc translocation and EGFR activation evolve differentially is in accordance with a time-dependent decoupling of EGFR activation and Shc translocation.
Concluding remarks
The subcellular analysis of EGFR-dependent signal transduction demonstrates that the earliest molecular events already generate heterogeneity in signal transduction in a time-dependent manner. While differences in receptor subpopulations may clearly contribute to this phenomenon, a time-dependent decoupling of the different components augments the effect. The observations made for EGFR-dependent signaling at the subcellular level have important implications for our general understanding of the underlying signal transduction mechanisms. Based on our observations, we propose a concept of signaling coherence to describe a signal transduction mechanism leading to a synchronized propagation of a signal at different subcellular locations or within a cell population. Our findings for the early steps in EGFR-dependent signal transduction are in contrast to the switch-like behavior of the downstream MAPK kinase cascade in this signaling pathway (Huang and Ferrell, 1996). For such a switch-like behavior, a sustained correlation as well as a decrease of the CVs would be expected. It would be worthwhile applying the method of focal EGFR stimulation (QMRA) to investigate whether subcellular heterogeneities also exist in the toggling of this switch. One is tempted to speculate that signaling coherence increases or even that activation states converge along a given signal transduction pathway. In the specific system we have addressed, the differential increase in the CVs revealed that the translocation of Shc apparently follows a more synchronized kinetic scheme than the preceding step(s) of EGFR activation.
In this work QMRA was employed for analysis of the kinetics of signal transduction at the subcellular level. This goal was achieved through the combination of local stimulation, preservation of cellular integrity, confocal immunofluorescence microscopy and three-dimensional image analysis. Further potential applications of QMRA include the simultaneous analysis of different signal transduction pathways applying sets of microspheres with different ligands, and the dissection of events leading to receptor activation and heterodimerizations with microspheres coated with anti-receptor monoclonal antibodies. Due to the rapid engulfment by plasma membrane and internalization, quantitative microsphere approaches are also applicable to questions for which internalization is relevant. Another interesting aspect concerns the identification of steps in a signal transduction pathway that distribute the signal throughout a cell. A microsphere-based approach for the study of integrin and cadherin signaling has been reported (Levenberg et al., 1998). The combination of QMRA with GFP fusion proteins, the strategy adopted in the present investigation, constitutes a general method for following the kinetics of protein activation and translocation at the subcellular level in vivo.
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ACKNOWLEDGMENTS |
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