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Lateral Propagation of EGF Signaling after Local Stimulation Is Dependent on Receptor Density
Developmental Cell, Vol. 3, 245–257, August, 2002, Copyright 2002 by Cell Press
Lateral Propagation of EGF Signaling after Local Stimulation Is Dependent on Receptor Density Asako Sawano,1,2 Shuichi Takayama,3 Michiyuki Matsuda,4 and Atsushi Miyawaki1,5 1 Laboratory for Cell Function and Dynamics Advanced Technology Development Center Brain Science Institute RIKEN 2-1 Hirosawa Wako-city, Saitama 351-0198 Japan 2 Brain Science Research Division Brain Science and Life Technology Research Foundation 1-28-12 Narimasu Itabashi, Tokyo 175-0094 Japan 3 Departments of Biomedical Engineering and Macromolecular Science and Engineering University of Michigan Ann Arbor, Michigan 48109 4 Department of Tumor Virology Research Institute for Microbial Diseases Osaka University 3-1 Yamadaoka Suita-shi, Osaka 565-0871 Japan
Summary We analyzed lateral propagation of epidermal growth factor (EGF) signaling in single live COS cells following local stimulation, achieved by the use of laminar flows containing rhodamine-labeled EGF. The spatiotemporal pattern of EGF signaling was visualized by fluorescent indicators for Ras activation and tyrosine phosphorylation. Contrary to the findings in previous reports, both signals were localized to the stimulated regions in control COS cells expressing EGF receptor at the basal level. However, the signals spread over the entire cell when EGF receptors were overexpressed or when receptor/ligand endocytosis was blocked. We thus present evidence that ligand-independent propagation of EGF signaling occurs only when the receptor density on the plasma membrane is high, such as in carcinoma cells. Introduction Signaling by ligand-activated receptor tyrosine kinases (RTKs), such as EGF receptor (EGFR), elicits a wide range of cell type-specific responses leading to proliferation, differentiation, apoptosis, and migration (reviewed in Hackel et al., 1999; Schlessinger, 2000). Upon ligand binding, RTKs dimerize and undergo autophosphorylation at specific tyrosine residues. In turn, these phosphotyrosines and their adjacent sequences specifically recruit signaling molecules via SH2 and PTB domains. The 5
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recruited molecules are adaptor proteins, such as Shc, Grb2, and CrkII, or enzymes, like PLC␥. They mediate subsequent activation of a variety of downstream signaling cascades. Indicators for various cellular events have been developed by a green fluorescent protein (GFP)-based fluorescence resonance energy transfer (FRET) technique (Miyawaki et al., 1997; Janetopoulos et al., 2001; Ting et al., 2001; Zaccolo et al., 2000; Zhang et al., 2001; van Roessel and Brand, 2002). We also generated novel fluorescent indicators to monitor activation of Ras family G proteins (Raichu-Ras) (Mochizuki et al., 2001) and tyrosine phosphorylation (Picchu-X) (Kurokawa et al., 2001). Raichu-Ras consists of H-Ras, the Ras binding domain (RBD) of Raf, and yellow- and cyan-emitting mutants of GFP, YFP, and CFP, respectively. Intramolecular interactions between active, GTP-bound Ras and the RBD bring YFP and CFP into close proximity, thereby increasing FRET. Picchu-X consists of CrkII sandwiched with YFP and CFP. Intramolecular binding of the SH2 domain to a phosphorylated tyrosine increases FRET. Using these probes, we were able to obtain spatiotemporal images of EGF-induced Ras activation and tyrosine phosphorylation in single living cells. Upon application of EGF to cultured COS cells, which brings about global activation of EGFRs, Ras activation was observed to occur at the peripheral plasma membrane and was reduced at sites of cell-cell contact (Mochizuki et al., 2001), while tyrosine phosphorylation of CrkII beneath the plasma membrane was distributed evenly throughout the cell (Kurokawa et al., 2001). In most cell-biological experiments, cultured cells are bathed in ligand solutions, whereas cells in intact tissues are stimulated locally in most physiological situations. It would be interesting to learn how EGF signaling spreads within a cell after local stimulation with EGF. While activation of EGFR is generally thought to activate the RasRaf-MEK-ERK pathway and thereby convey information to the nucleus, evidence has accumulated that EGF signaling is involved in many other spatially dynamic cellular processes, such as membrane trafficking (Barbieri et al., 1998), turnover of focal adhesions (Schlaepfer and Hunter, 1998), and cytoskeletal organization (Ho and Bretscher, 2001). Therefore, it is important to understand whether the EGF signaling remains localized or is propagated in response to local stimulation. Verveer et al. (2000) reported a new signaling mechanism described as ligand-independent lateral propagation of receptor phosphorylation in the plasma membrane and suggested a dynamic equilibrium between monomeric and dimeric activated EGFR. The authors elegantly monitored EGFR phosphorylation by measuring FRET between GFP fused to the carboxyl terminus of the receptor and Cy3 attached to a phosphotyrosinespecific antibody. They observed rapid and extensive propagation of receptor phosphorylation over the entire cell (MCF7 cell) after local stimulation with EGF covalently attached to beads. In order to observe these cellular events, the investigators needed to introduce a large number of EGFRs into the cells, though this approach
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Figure 1. Visualization of Rhodamine-EGFEGFR Complexes Time-lapsed images of A431 cells (A and B), normal COS cells (C and D), and COS cells transfected with the cDNA for EGFR (E and F) when incubated with 50 ng/ml rhodaminelabeled EGF. The fluorescence signals were monitored every 10 s through the rhodamine channel. Images taken after 10 s (A, C, and E) and 20 min (B, D, and F) of incubation are presented. For estimation of EGFR plasma membrane expression level, images (C) and (E) were taken under the same optical conditions and are displayed using the same grayscale. In contrast, image (A) was captured with 20-fold-less sensitivity. (E) EGFR-overproducing COS cells and untransfected COS cells are indicated by asterisks and arrowheads, respectively. Scale bar, 10 m.
is known to make the observed cells cancerous. In such pathological situations, we also observed that local stimulation with EGF caused rapid and global propagation of Ras activation and tyrosine phosphorylation beneath the plasma membrane. Though the ligand-independent propagation mechanism initially proposed was understood to be at work in all cell types, we have argued against generalizing the results of the aforementioned experiments to suggest that this is a common physiological mechanism. In fact, here, we offer the first evidence that EGF signaling remains localized in cells that express EGFR at a basal level. A more comprehensive view of EGF signaling pattern must include the understanding that EGF-independent signal propagation depends on the density of EGFRs in the plasma membrane, which is, in turn, related to the saturable endocytosis (Wiley, 1988) of EGF-EGFR complexes. Results Visualization of EGF-EGFR Complex Dynamics in Membranes We followed the fate of rhodamine-labeled EGF molecules that were applied over A431 (human epidermoid carcinoma cell line) and COS cells. The ligands were added in Hank’s Balanced Salt Solution (HBSS) medium at a final concentration of 50 ng/ml. Immediately after application (10 s), the A431 cells displayed heavy labeling of their plasma membranes (Figure 1A), suggesting
high receptor density on these cells. At later time points (20 min), the A431 cells were found to have ineffectively internalized this high level of occupied EGFR, apparently because of a limited amount of a specific component responsible for mediating EGF-induced receptor internalization (Wiley, 1988). While a punctate pattern of fluorescence consistent with internalization was observed, a significant amount of rhodamine-EGF remained on the plasma membrane (Figure 1B). It was also noted that the EGF-stimulation induced refractile changes and detachment from the substratum. COS cells were treated with rhodamine-EGF in the same manner, and fluorescence images (Figures 1C–1F) were captured. These images required a longer exposure time than did the A431 cell images because of the comparatively low receptor expression of the COS cells. The ligands were again observed to be bound to EGFRs on the plasma membrane (Figure 1C), though quantitative analysis indicated an approximately 50-fold difference in the absolute signal intensity between COS and A431 cells. The EGFR densities on A431 and COS cells were previously reported to be roughly 2 ⫻ 106 and 4 ⫻ 104 molecules per cell, respectively (Wiley, 1988; Livneh et al., 1986). Assuming that a linear correlation between plasma membrane labeling and the actual number of receptors exists, it would then be possible to estimate roughly the receptor density from the plasma membrane fluorescence intensity. By 20 min after stimulation, the fluorescent labeling on the plasma membrane of the
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COS cells had disappeared, and punctate signals had emerged at the periphery and in the perinuclear region (Figure 1D), indicating that most of the EGF-EGFR complexes had been internalized into endosomal compartments. EGFR internalization was also visualized in COS cells by immunocytochemical analysis with an antiEGFR antibody (data not shown). The resulting immunosignals displayed similar patterns to the rhodamineEGF signals. Thus, the rhodamine-EGF image can be viewed as roughly reflecting the distribution of EGFR, in spite of the segregation of ligand and receptor at late stages of endocytosis (Carter and Sorkin, 1998). When COS cells were transfected with EGFR cDNA, a portion of the cells produced a large quantity of receptors. Ten seconds after stimulation with rhodamine-EGF, the plasma membranes of the transfected cells (Figure 1E, asterisks) were intensely labeled. Later, these EGFRoverexpressing cells also displayed saturated EGFEGFR endocytosis (Figure 1F), as did the A431 cells from the previous experiment. The fluorescence-signal intensities of the cells in Figures 1C and 1E were comparable, since the two images were taken under the same conditions. The EGFR density of the transfected cells was estimated to be around 5 ⫻ 105 molecules per cell. Clearly, the cells indicated by arrowheads in Figure 1E were untransfected, as they exhibited the same rhodamine-EGF labeling as the control cells in Figures 1C and 1D. Local Stimulation by the Use of Laminar Flow Figure 2A is a diagram of the microfluidic channel that was used to subject cells to local stimulation (Takayama et al., 1999, 2001). It has three inlets and one outlet. The optimal channel size for our COS cell/EGF experiments was attained with 150 m-wide rectangular capillaries. The performance of the channel with confluent COS cells (Figure 2B) was examined with fluorescent EGF molecules. FITC- and rhodamine-labeled EGF were made to flow over the COS cells from the left and right inlets, respectively, together with medium from the middle inlet. A merged green and red fluorescence image is shown in Figure 2C. Stable laminar streams were always maintained for over one hour. We found that a submaximal dose of EGF gave rise to a graded amplitude of Raichu-Ras signal, while a maximal dose increased the amplitude to a saturated level. In culture dishes, cells are exposed to EGF ligands in the medium, and it is by this system that most previous studies regarding EGF signaling have been carried out. In our microfluidic channels, on the other hand, cells are continuously exposed to moving EGF ligands in the flow stream. For this reason, we decided to examine the relationships between EGF concentration and the amplitude of Raichu-Ras signal in normal COS cells using both experimental systems. The dose-response curve obtained with the channel was found to be shifted slightly to the right (Figure 2D). Local Activation of Ras in Normal COS Cells We analyzed the spatiotemporal pattern of Ras activation in response to a laminar flow in a normal COS cell plated in the microfluidic channel (Figure 3). The cell
that expressed Raichu-Ras protein was stimulated locally with a solution containing 50 ng/ml of rhodamineEGF. This concentration of EGF ligands should be saturating within the stimulated region (see Figure 2D). A fluorescence image of the cell was taken through the FRET channel (440 ⫾ 10 nm excitation, 535 ⫾ 12.5 nm emission) (Figure 3A). Rhodamine-EGF flowed over the right-hand section, as indicated by violet shading. To confirm the presence of a stable, controlled laminar flow, we monitored the red fluorescence of the bound rhodamine-EGF through the rhodamine channel (546 ⫾ 5 nm excitation, 595 ⫾ 30 nm emission). After stimulation for 10 min, the area to which the ligands bound was visualized, including the neighboring cells that did not express Raichu-Ras protein (Figure 3B). The focal plane was on the top plasma membrane, and, therefore, the punctate fluorescence pattern characteristic of endocytosis is not seen. Internalization of EGF-EGFR complexes, however, was visualized by viewing a different focal plane (data not shown). Figure 3D shows representative pseudocolored emission-ratio images of Raichu-Ras, which were made by assigning red and blue to high and low levels of Ras activation, respectively. In addition, the temporal profiles of the ratio values in the regions of interest (ROIs) indicated in Figure 3A are shown (Figure 3H). Ras activation increased and reached a maximum within 10 min in the region that was exposed to rhodamine-EGF. In contrast, little or no activation was seen in the unexposed parts of the cell. After 10 min, a noticeable similarity between the patterns of rhodamine-EGF binding (Figure 3B) and Ras activation (Figure 3D) was observed. The outlines of the cell at 0 and 20 min are superimposed (Figure 3C). Although this COS cell was surrounded by cells on every side, the spatially restricted stimulation resulted in a slight, but definite, morphological change that was localized to the stimulated region. Such morphological change occasionally affected the flow pattern, resulting in an indistinct leak of EGF ligands. Thus, Raichu-Ras signal was observed to ooze slightly out of the stimulated region (Figure 3D). But the leaking signal was only transient and never crossed the cell at a later time point (⬎1 hr). Another normal COS cell did not show any morphological change with the rhodamine-EGF flow over almost half the area of the cell (Figures 3E and 3F). The Ras signal reached the maximum at 10 min and then gradually faded out (Figures 3G and 3I). Similar experiments were performed using 11 different normal COS cells. In all cases, local stimulation with EGF resulted in localized activation of Ras (Table 1). Localized Tyrosine Phosphorylation in Normal COS Cells Next we observed tyrosine phosphorylation events in a normal COS cell expressing Picchu-X (Figure 4A), which, again, employs FRET between YFP and CFP (Kurokawa et al., 2001). This in vivo probe has the CAAX box of KiRas at its carboxyl terminus. This modification restricts its localization to the plasma membrane, thereby improving the sensitivity and spatial resolution of the system for detecting tyrosine phosphorylation. This plasma membrane localization also suggests that the Picchu-X signal may reflect the phosphorylation status of EGFR. In fact, the requirement of EGFR kinase activity for the
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Figure 2. Microfluidic Channel for Laminar Flow Experiments (A) Top view of the capillary network. (B) A bright-field micrograph showing the junction where the three inlets converge. The channel contained confluent COS cells. A 20⫻ objective lens was used. (C) Fluorescence micrograph of the same field as in (B). The FITC-EGF and rhodamineEGF fluorescence images were captured separately and merged. Scale bar, 100 m. (D) Dose-response curves for EGF-induced Ras activation in normal COS cells expressing Raichu-Ras. The EGF-induced increase in the emission ratio of Raichu-Ras (R/R0; the relative difference of the observed ratio [R] and the prestimulus value [R0]) was measured in culture dish (dotted line) and in the microfluidic channel (solid line). The saturating level of R/R0 ranged from 1.2 to 1.4. Each point is the mean ⫾ SD of three to six experiments.
Picchu-X signal was confirmed pharmacologically (Kurokawa et al., 2001). Consistent with the view that Picchu-X is an early detector of EGF signaling, its emission ratio started to increase immediately and reached its maximum within 1 min after the addition of EGF (Figure 4D). Like the Ras activation shown in Figure 3, tyrosine phosphorylation signals were localized to the same portion of the cell as the EGF stimulation (Figures 4C and 4D), where rhodamine-EGF was bound (Figure 4B). There was no observable tyrosine phosphorylation signal in the left part containing ROIs 3 and 4 , even after 25 min of stimulation. Subsequently, 50 ng/ml rhodamineEGF were added to the left stream to stimulate the nonresponding region. An immediate increase in the emission ratio was observed (Figure 4E), indicating that the EGFRs in the left region had not been desensitized. Localized Versus Propagated EGF Signaling Our results mentioned above seemed to contradict the previous report by Verveer et al. (2000), which concluded that the phosphorylation of EGFR was propagated quickly after focal stimulation with EGF beads. There were, however, a couple of differences between the two experimental protocols. In their experiments, first, EGFR-GFP chimeric proteins were highly expressed in MCF7 carcinoma cells to allow monitoring the phosphorylation of the exogenous EGFR. The density of
EGFRs on the cells would have then been remarkably high (5 ⫻ 105 per cell) compared with normal COS cells. Second, the cells were focally stimulated using 0.8 mdiameter beads conjugated with EGF molecules. Although the ability of these beads to activate EGFR and the subsequent internalization of the receptor-ligand complexes have been shown (Verveer et al., 2000; Brock and Jovin, 2001), artifacts due to this mode of EGF delivery cannot be disregarded. We hypothesized that the dynamics of EGFR activation could depend on the density of receptors in the plasma membrane. To test this hypothesis, we increased the receptor density by expressing EGFR in normal COS cells. Also, since endocytosis of ligandreceptor complexes is closely related to the receptor density and is important for regulation of signaling, we opted to use the soluble form EGF to allow physiological endocytosis to occur. Spatial Propagation of EGF Signaling in Cells Overexpressing EGFR COS cells in the microfluidic channel were cotransfected with cDNAs for Raichu-Ras and full-length EGFR. EGFR and Raichu-Ras were expressed with various ratios in the transfected cells. The Raichu-Ras-expressing cell shown in Figure 5A expressed a significant amount of EGFR. This cell was next to another that displayed a
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Figure 3. Local Stimulation of Normal COS Cells with Rhodamine-EGF for Visualization of Ras Activation (A) Fluorescence image of Raichu-Ras before stimulation. The region of rhodamine-EGF exposure is shaded in violet. Across the laminar flows, four ROIs are assigned. Scale bar, 10 m. (B) Rhodamine-EGF fluorescence after 10 min stimulation. The outline of the COS cell is shown by a dotted line. (C) Superimposed outlines of the cell at 0 (black) and 20 (red) min. (D) A series of dual-emission ratio (535 ⫾ 12.5 nm to 480 ⫾ 15 nm) images presented in intensity-modified display (IMD) mode. Time points (min) after initiation of stimulation are shown in the top right corners. (E) Fluorescence image of Raichu-Ras in another normal COS cell before stimulation with rhodamine-EGF (violet). Four ROIs are assigned as in (A). Scale bar, 10 m. (F) Rhodamine-EGF fluorescence after 20 min stimulation in the COS cell shown in (E), which is outlined by a dotted line. The lens was focused to see the punctate fluorescence pattern characteristic of endocytosis. (G) A series of dual-emission ratio images from the COS cell shown in (E), presented in a manner similar to that in (D). (H and I) Temporal profiles of the ratios in the ROIs indicated in (A) and (E), respectively. The changes in ratio were normalized to the initial ratio values. Red circles and squares are the data points from the stimulated side: ROIs 1 and 2, respectively. Blue circles and squares are from the unstimulated side: ROIs 3 and 4, respectively.
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Table 1. EGFR Expression Dependency of EGF Signaling Propagation Indicators
EGF Binding Sites/Cell
Raichu-Ras
3–5 ⫻ 104 3–5 ⫻ 104
⫹EGFR
Picchu-X ⫹EGFR
Propagation/Number of Experiments ⫹MDC
1–3 ⫻ 105 3–6 ⫻ 105 6–8 ⫻ 105 3–5 ⫻ 104 3–5 ⫻ 104 1–3 ⫻ 105 3–6 ⫻ 105
0/13 3/3 3/3 4/4 2/2
⫹MDC
0/8 2/2 1/1 4/4
The results of 40 experiments are summarized. In every experiment, the observed cells were mostly confluent and were stimulated over 10–50% of their surface areas.
similar level of EGFR expression, yet did not express Raichu-Ras (Figure 5B, arrowhead). The arrow in Figure 5B indicates the presence of cells that produced neither Raichu-Ras nor exogenous EGFR; the fluorescence signal from the rhodamine-EGF on untransfected COS cells was barely visible because of the short exposure time optimized for the high levels of EGFR on the transfected cells. From the intensity of the red plasma membrane fluorescence, the cell of interest was estimated to contain roughly 4 ⫻ 105 binding sites. After 1 min of local stimulation, the Raichu-Ras signal was noticeable across the entire cell and reached a maximum within 10 min in both the stimulated and unstimulated regions (Figures 5C and 5G). It should be noted that the majority of rhodamine-EGF-EGFR complexes remained in the plasma membrane because of saturation of endocytosis (Figures 1E, 1F, and 5B). Such global activation of Ras was observed in each of eight other COS cells that overexpressed EGFR in a range from 1 ⫻ 105 to 8 ⫻ 105 molecules per cell (Table 1). Next, another COS cell overexpressing EGFR (8 ⫻ 105 binding sites) was locally stimulated with a submaximal dose of rhodamine-EGF (1 ng/ml) (Figures 5D and 5E). The Raichu-Ras signal increased to a submaximal level, evenly throughout the cell (Figures 5F and 5H). The subsequent application of 50 ng/ml rhodamine-EGF then caused a further increase in the signal to a saturating level (Figure 5H). The ligand-independent propagation of EGFR phosphorylation (or activation) after focal stimulation (Verveer et al., 2000) was substantially reproduced in our experiment that employed a COS cell expressing Picchu-X and a high number of EGFRs (3 ⫻ 105 binding sites per cell) (Figure 6). Figures 6C and 6D show that local stimulation (Figures 6A and 6B) for 3 min resulted in a global increase in the emission ratio of Picchu-X, suggesting full activation of EGFRs throughout the cell. It should be noted that we observed this spread of tyrosine phosphorylation events in a single live COS cell in real time, while the previous study used many different cells that were fixed at various time points and reacted with anti-phosphotyrosine antibody. Mechanisms for Localized EGF (Ras) Signaling We studied further the mechanisms underlying the localized activation of Ras in normal COS cells, as presented
in Figure 3. First, we examined whether endocytosis of EGF-EGFR complexes negatively regulated the propagation of Ras activation. COS cells in the microfluidic channel were pretreated with 20 M monodansylcadaverine (MDC), an inhibitor of clathrin-dependent endocytosis, for 10 min. Then, 50 ng/ml of rhodamine-EGF were applied to the left part of a COS cell expressing RaichuRas (Figure 7A), whereupon the plasma membrane on that side became homogeneously labeled, though some bright spots were also detected (Figure 7B). No punctate fluorescence pattern was observed in different focal planes. The surface labeling lasted for over 20 min, indicating that the blockade of ligand-receptor endocytosis had occurred. The resulting accumulation of EGF-EGFR complexes in the plasma membrane resulted in gradual, but global, activation of Ras within the entire cell (Figure 7C). The emission ratio of Raichu-Ras increased by about 40% after 20 min. Importantly, this Ras activation signal was specifically due to the EGF stimulation. In comparison with the strong Ras signal observed in the tested cell, the increase in the emission ratio in adjacent cells (Figure 7C, arrowheads), which had also been treated with MDC, was negligible, at only 1% of the basal level. Gradual propagation of tyrosine phosphorylation was observed also in Picchu-X-expressing COS cells that had been treated with MDC (Table 1). These results support the idea that EGFRs in normal COS cells are effectively downregulated by endocytosis to prevent lateral propagation of EGF signaling. Similar global delocalization of Ras activation and tyrosine phosphorylation was observed in normal COS cells expressing a moderate amount of an oncogenic version of c-Cbl (70ZCbl) (Levkowitz et al., 1999; Lill et al., 2000; Soubeyran et al., 2002), which recent results argue prevents endocytosis. Next, we examined the activation of another Ras family G protein, Rap1, which is activated inside cells by EGF and in a manner dependent on endocytosis. A COS cell expressing Raichu-Rap1 (Mochizuki et al., 2001) was locally stimulated in the microfluidic channel (Figure 7D). After 20 min of stimulation, EGF-EGFR complexes were mostly internalized, but localized within the stimulated region (Figure 7E). Activation of Rap1, however, was initiated in the perinuclear region and spread in an isotropic manner toward the plasma membrane (Figure
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Figure 4. Local Stimulation of a Normal COS Cell with Rhodamine-EGF for Visualization of Tyrosine Phosphorylation beneath the Plasma Membrane (A) Fluorescence image of Picchu-X with four ROIs; the rhodamine-EGF flow is shaded in violet. Scale bar, 10 m. (B) Rhodamine-EGF fluorescence images after 10 and 20 min stimulation, with the outline of the COS cell depicted by a dotted line. Over the monolayer culture is a round cell, which was intensely labeled (indicated by an asterisk). (C) A series of dual-emission ratio images presented in IMD mode. Time points (min) after the start of stimulation are shown in the top right corners. (D) Temporal profiles of the ratios in the ROIs indicated in (A). Data points are plotted as in Figures 3H and 3I. (E) At 30 min, a ratio image was captured after global stimulation with rhodamine-EGF.
7F). This observation suggests that the guanine nucleotide exchange factor for Rap1 may be activated not only by the internalized EGFRs, but also by diffusible messenger molecules, and that the activation of Rap 1 outside the stimulated region may block the propagation of Ras-dependent pathways inside the cells. It is interesting that activation of Ras, but not Rap1, retains the spatial information regarding the source of EGF signaling in the plasma membrane. Discussion When a cell is stimulated locally with ligands, is the activation of intracellular signaling localized or propagated over the entire cell? To address this intriguing and fundamental question, we investigated the spatiotemporal pattern of EGF signaling in single live COS
cells. A summary of our results is presented in Table 1. We demonstrate that ligand-independent lateral signal propagation depends on the plasma membrane receptor density, helping to explain the previous findings reported on this subject. To obtain a better understanding of the regulation of signaling dynamics, we also employed the following three technologies recently developed in our laboratories: (1) Raichu-Ras (Mochizuki et al., 2001) and Picchu-X (Kurokawa et al., 2001), fluorescent indicators that can pinpoint the site of Ras activation and tyrosine phosphorylation beneath the plasma membrane, respectively, within single live cells; (2) microfluidic systems, which create controlled, stable laminar flow to make possible finely localized treatment of cells with soluble ligands (Takayama et al., 1999, 2001); (3) an advanced multicolor imaging system for conventional fluorescence microscopy (Sawano et al., 2002).
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Figure 5. Local Stimulation of EGFR-Overproducing COS Cells with Rhodamine-EGF for Visualization of Ras Activation (A) Fluorescence image of Raichu-Ras in a COS cell transfected with EGFR cDNA. The cell was nearly in contact with the left wall of the capillary. Four ROIs and the rhodamine-EGF flow (violet) are shown. Scale bar, 10 m. (B) Rhodamine-EGF fluorescence after 10 min stimulation. The dotted line depicts the outline of the COS cell. A neighboring cell, which produced a large quantity of EGFRs, is indicated by an arrowhead. An arrow defines other neighboring cells that were not transfected. This image was taken with 10-fold-less sensitivity than the images in Figures 3B and 4B. (C) A series of dual-emission ratio images presented in IMD mode. Time points (min) after initiation of stimulation are shown in the top right corners. (D) Fluorescence image of Raichu-Ras in another EGFR-overexpressing COS cell before stimulation with rhodamine-EGF (violet). Four ROIs are assigned as in (A). Scale bar, 10 m. (E) Rhodamine-EGF fluorescence after 10 min stimulation in the COS cell shown in (D), which is outlined by a dotted line. (F) A series of dual-emission ratio images from the COS cell shown in (D), presented in a manner similar to that in (C). (G and H) Temporal profiles of the ratios in the ROIs indicated in (A) and (D), respectively. Data points are plotted as in Figures 3H and 3I. Subsequent addition of 50 ng/ml rhodamine-EGF is indicated by an arrowhead in (H).
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Figure 6. Local Stimulation of an EGFROverproducing COS Cell with RhodamineEGF for Visualization of Tyrosine Phosphorylation beneath the Plasma Membrane (A) Fluorescence image of Picchu-X with four ROIs, with the rhodamine-EGF flow shaded in violet. Scale bar, 10 m. (B) Rhodamine-EGF fluorescence images after 10 and 20 min stimulation. The outline of the COS cell is demarcated by a dotted line. The camera settings were the same as those for the image in Figure 5B. (C) A series of dual-emission ratio images presented in IMD mode. Time points (min) after initiation of stimulation are shown in the top right corners. (D) Temporal profiles of the ratios in the ROIs indicated in (A). Data points are plotted as in Figures 3H and 3I.
Previous studies of EGF signaling have been performed using cells that express high levels of EGFRs. In one study, GFP variants were fused to the carboxyl terminus of the EGFR. The functionality of the EGFRGFP was verified (Carter and Sorkin, 1998), and the proteins were used to monitor phosphorylation (Verveer et al., 2000) and endocytosis of the EGFR (Carter and Sorkin, 1998) and the interaction between the EGFR and adaptor proteins (Sorkin et al., 2000). Expression of EGFR-GFP, however, results in a high density of functional receptors on the plasma membrane, creating a situation where receptor activation spreads via dynamic dissociation-association equilibrium, as proposed by Verveer et al. (2000). Therefore, we avoided the introduction of any GFP-labeled EGFR as an indicator. Instead, we focused on visualizing two EGF-dependent events: Ras activation and tyrosine phosphorylation beneath the plasma membrane. Another study used A431 carcinoma cells, which have an extraordinarily high number of EGFR molecules (1–3 ⫻ 106 per cell) (Wiley, 1988). It is reasonable to assume that, according to the above
argument, EGFR dimers (oligomers) are formed before the 1:1 interaction with EGF, as revealed by single-molecule imaging (Sako et al., 2000) or FRET analysis (Gadella and Jovin, 1995). However, because of autocrine regulation of A431 cells through production of transforming growth factor ␣ (TGF-␣) (Harder et al., 1998), it would be difficult to attribute changes to local stimulation with EGF. Also, because the cells exhibited refractile changes and detachment from the substratum upon EGF treatment (Figures 1A and 1B), we concluded that these cells were inappropriate for our imaging experiments with laminar flows. As a result, we chose to use COS cells, which usually express a small number of EGFRs (3–5 ⫻ 104 per cell) (Livneh et al., 1986). Overexpression of cDNA-derived EGFR greatly increased the receptor density on the surface of the cells. Transfected cells that displayed a moderately high number of EGFRs (1–8 ⫻ 105 per cell) and that were adhesive to the substratum were used for imaging experiments. A comparative study using normal and transfected COS cells was performed to give us a
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Figure 7. Propagation of Ras Activation after Blockade of Endocytosis and Visualization of Rap1 Activation in Normal COS Cells (A) Fluorescence image of Raichu-Ras in a COS cell after treatment with MDC but before stimulation. This cell was neighbored by a few other cells on the right side, as indicated by arrowheads (C). The rhodamine-EGF flow (violet) is shown. Scale bar, 10 m. (B) Rhodamine-EGF fluorescence images after 10 and 20 min stimulation. The outline of the COS cell is shown by a dotted line. (C) A series of dual-emission ratio images for Ras activation presented in IMD mode. Time points (min) after initiation of stimulation are shown in the top right corners. After 20 min, no activation of Ras was observed in the neighboring cells (arrowheads), which had also been treated with MDC. (D) Fluorescence image of Raichu-Rap1 in a COS cell with the rhodamine-EGF flow shaded in violet. Scale bar, 10 m. (E) Rhodamine-EGF fluorescence images captured after 10 and 20 min stimulation. The outline of the COS cell is shown by a dotted line. (F) A series of dual-emission ratio images of Rap1 activation presented in IMD mode. Time points (min) after initiation of stimulation are shown in the top right corners.
direct answer to the question of how EGFR density affects the propagation of receptor activation and its downstream events. The central finding in our study is that, in normal COS cells, local stimulation with a maximal dose (50 ng/ml) of rhodamine-EGF led to spatially restricted EGF signaling. It should be noted that the saturable activation of EGFR in a local region effectively confines the downstream signaling. We previously reported the sustained activation of Ras for over 10 hr in the neurites of PC12 cells expressing Raichu-Ras (Mochizuki et al., 2001). Since the fluorescence intensity of PC12 neurites expressing YFPRas recovered within 4 min after photobleaching, it was concluded that the local activity of Ras is determined by a local balance between the activity of guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP), rather than by the retention of activated Raichu-Ras (Mochizuki et al., 2001). The localization of the
signals of Raichu-Ras and Picchu-X in the present study also suggests that the indicators have rapid kinetics of activation and deactivation. The internalization of activated receptors is generally viewed as a mechanism to attenuate signaling. Activation of EGFRs by ligands also triggers rapid endocytosis of EGF-EGFR complexes. Although it has been reported that certain pathways mediated by EGFR are activated in endosomes (Sorkin and Carpenter, 1991; Haugh et al., 1999), the internalized EGFR is mostly ubiquitinated and then degraded in proteosomes (Waterman et al., 2002). This process is controlled by complex formation of Cbl with CIN85-endophillin (Soubeyran et al., 2002; Petrelli et al., 2002). Another recent study has shown that the endocytosed EGFR is efficiently inactivated through dephosphorylation by protein tyrosine phosphatase (PTP) 1B on the surface of the endoplasmic reticulum before degradation (Haj et al., 2002). In either case, the
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endocytosis plays a pivotal role in the inactivation of EGFR. Although small beads conjugated with EGF molecules have often been used for focal stimulation and have been observed to be taken up by cells (Verveer et al., 2000; Brock and Jovin, 2001), they may impede normal physiological internalization and metabolism of the EGF-EGFR complexes. Thus, we studied the internalization of ligand-receptor complexes using rhodaminelabeled EGF for stimulation. The pH-resistant fluorescence of rhodamine permitted quantitative monitoring in acidic organelles, such as endosomes, though segregation of ligand and receptor does occur at these late stages. We reproduced the results of Figures 3–7 using unlabeled EGF (data not shown), to prove that rhodamine conjugation does not affect its interaction with EGFRs. How is endocytosis involved in the spatiotemporal pattern of EGF signaling that we observed in this study? When EGFR-overexpressing COS cells were stimulated, a large number of EGFRs remained in the membrane well after stimulation, most likely due to saturation of EGF-EGFR endocytosis. This situation probably facilitated transient dimer formation of EGFR in the plasma membrane, resulting in the observed ligand-independent propagation of receptor activation. In normal COS cells, by contrast, the EGFRs are efficiently cleared off the plasma membrane soon after stimulation, allowing for localized receptor activation. Thus, when EGF-EGFR endocytosis was blocked, the receptor activation was gradually delocalized. Within a cell, on the other hand, diffusible factors may work to augment the propagation of receptor activation. Recent evidence has shown that activation of EGFR results in the generation of hydrogen peroxide (H2O2), which upregulates tyrosine phosphorylation signaling by inhibiting PTP activity (Rhee et al., 2000). It is unlikely that the internalized activated EGFRs contributed to the Raichu-Ras and Picchu-X signals observed in our study because the activation of both indicators is exclusively localized to the plasma membrane (Mochizuki et al., 2001; Kurokawa et al., 2001). Even if the internalized EGFRs act on the nearby plasma membrane, they are most likely not involved in the lateral propagation of EGF signaling shown in Figures 5 and 6. When EGFR was overexpressed, internalization of the ligand-receptor complexes was localized to the stimulated side, and the red fluorescence did not spread to the opposite side of the cell (see Figure 6B). There are many interesting biological consequences of the observed local and global activation of EGF signaling. One of the well-studied downstream signaling targets of EGFR is the Ras-Raf-MEK-ERK pathway. Since this pathway converges on the nucleus, the spatial information pertaining to the site of EGFR activation is likely to be lost. In contrast, other EGFR-dependent pathways, such as membrane trafficking, turnover of focal adhesions, and cytoskeleton organization, are spatially restricted, contributing to directional cell morphology and motility. In our experiments, the localized spatial pattern of EGF signaling was well correlated with local morphological changes (Figure 3C), potentially involving not only the Ras pathway, but also other EGF-induced signaling molecules, such as Rho, focal adhesion kinase (Lu et al., 2001), and phosphatidylinositol-3 kinase (Arcaro et al., 2000). In organogenesis, local activation of
EGF signaling appears to be important for coordinated control of region-specific detachment from the extracellular matrix. This process may allow for proliferation of cells with regulated EGFR expression while maintaining architectural order. On the other hand, a variety of carcinoma cells overexpress EGFR and show dysregulated cell motility upon EGF treatment, as do A431 cells. Ligand-independent propagation of EGF signaling may be one of the molecular mechanisms underlying invasion and metastasis, criteria by which malignant tumors are characterized. Experimental Procedures Microfluidic Channels Polydimethylsiloxane (PDMS) slabs with channel features were made as previously described (Takayama et al., 1999). Capillary channels were formed by placing the PDMS membrane on a cover glass that had been coated with 0.25% Poly-L-Lysine (Sigma) for 5 hr. Rhodamine-EGF and FITC-EGF were purchased from Molecular Probes. Cell Cultures COS cells and A431 carcinoma cells were maintained in Dulbecco’s modified eagle’s medium (DMEM) containing 10% fetal bovine serum, antibiotics, and glutamine (growth medium) in a 5% CO2 incubator at 37⬚C. For delivery of COS cells into the capillary channels, the outlet reservoir was filled with a suspension of cells (about 105 cells per milliliter in the growth medium). The cells were allowed to flow slowly into the main channel and adhere to the cover glass. Nonadherent cells were removed by washing. The cells were then incubated in 5% CO2 at 37⬚C for 1 day before cDNA transfection. Transfection Cells were transfected with pRaichu-Ras (Mochizuki et al., 2001), pPicchu-X (Kurokawa et al., 2001), pRaichu-Rap1 (Mochizuki et al., 2001), or pKU-EGFR (Gotoh et al., 1997) for expression of EGFR, by the Lipofectin method (Gibco BRL). COS cells in the channel were treated with the transfection solution in the presence of 5% CO2 at 37⬚C for 16 hr. After washing, the capillary system was filled with growth medium and incubated for another 1 or 2 days. Imaging For imaging experiments, the growth medium in the capillary system was replaced by HBSS buffer. Cells were imaged on an inverted microscope (Olympus IX70) with a standard 75 W xenon lamp, a 40⫻ objective lens (Uapo/340, N.A. 1.35), and a dichroic mirror (455 nm long pass). Interference filters (excitation and emission filters) set in wheels were mechanically automated using Lambda 10-2 hardware (Sutter Instruments, Novato, CA). Emitted light was captured with a cooled CCD camera (Cool Snap fx; Roper Scientific, Tucson, AZ). The whole system was controlled using MetaFluor 4.6.6 software (Universal Imaging, Media, PA). Raichu and Picchu-X both have cyan and yellow fluorescent proteins as donor and acceptor, respectively. For dual-emission ratio imaging, fluorescence pictures were captured alternately through the donor channel (440 ⫾ 10 nm excitation, 480 ⫾ 15 nm emission) and FRET channel (440 ⫾ 10 nm excitation, 535 ⫾ 12.5 nm emission). Rhodamine-EGF was imaged through the rhodamine channel (546 ⫾ 5 nm excitation, 595 ⫾ 30 nm emission). All fluorescence images were taken using the 455 nm-long pass dichroic mirror. About 5% of the total light at 546 nm was reflected by the dichroic mirror and was used for excitation of rhodamine (Sawano et al., 2002). Through the donor and FRET channel, transfected cells that expressed a moderate amount of the fluorescent indicator and were located close to the capillary junction were selected. A solution of rhodamine-EGF was then substituted into either the left or right inlet, so that the solution flowed over one of the edges of the selected cell. For blocking of endocytosis, the three inlets were filled with HBSS buffer containing 20 M monodansylcadaverine (MDC) (Calbiochem) for 10 min.
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Acknowledgments The authors would like to thank Xiaoyue Zhu, Takako Kogure, and Dr. Takashi Fukano for their assistance, Dr. Hamid Band for the plasmid encoding c-Cbl (70Z-Cbl), and Dr. David A. Zacharias for critical comments. This work was partly supported by grants from CREST of JST (Japan Science and Technology) and the Japanese Ministry of Education, Science and Technology. Received: April 17, 2002 Revised: June 14, 2002 References Arcaro, A., Zvelebil, M.J., Wallasch, C., Ullrich, A., Waterfield, M.D., and Domin, J. (2000). Class II phosphoinositide 3-kinases are downstream targets of activated poplypeptide growth factor receptors. Mol. Cell. Biol. 20, 3817–3830. Barbieri, M.A., Kohn, A.D., Roth, R.A., and Stahl, P.D. (1998). Protein kinase B/akt and Rab5 mediate Ras activation of endocytosis. J. Biol. Chem. 273, 19367–19370. Brock, R., and Jovin, T.M. (2001). Heterogeneity of signal transduction at the subcellular level: microsphere-based focal EGF receptor activation and stimulation of Shc translocation. J. Cell Sci. 114, 2437–2447. Carter, R.E., and Sorkin, A. (1998). Endocytosis of functional epidermal growth factor receptor-green fluorescent protein chimera. J. Biol. Chem. 273, 35000–35007. Gadella, T.W., and Jovin, T.M. (1995). Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation. J. Cell Biol. 129, 1543–1558. Gotoh, N., Toyoda, M., and Shibuya, M. (1997). Tyrosine phosphorylation sites at amino acids 239 and 240 of Shc are involved in epidermal growth factor-induced mitogenic signalling that is distinct from Ras/mitogen-activated protein kinase activation. Mol. Cell. Biol. 17, 1824–1831.
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Lateral Propagation of EGF Signaling after Local Stimulation Is Dependent on Receptor Density Asako Sawano,1,2 Shuichi Takayama,3 Michiyuki Matsuda,4 and Atsushi Miyawaki1,5 1 Laboratory for Cell Function and Dynamics Advanced Technology Development Center Brain Science Institute RIKEN 2-1 Hirosawa Wako-city, Saitama 351-0198 Japan 2 Brain Science Research Division Brain Science and Life Technology Research Foundation 1-28-12 Narimasu Itabashi, Tokyo 175-0094 Japan 3 Departments of Biomedical Engineering and Macromolecular Science and Engineering University of Michigan Ann Arbor, Michigan 48109 4 Department of Tumor Virology Research Institute for Microbial Diseases Osaka University 3-1 Yamadaoka Suita-shi, Osaka 565-0871 Japan
Summary We analyzed lateral propagation of epidermal growth factor (EGF) signaling in single live COS cells following local stimulation, achieved by the use of laminar flows containing rhodamine-labeled EGF. The spatiotemporal pattern of EGF signaling was visualized by fluorescent indicators for Ras activation and tyrosine phosphorylation. Contrary to the findings in previous reports, both signals were localized to the stimulated regions in control COS cells expressing EGF receptor at the basal level. However, the signals spread over the entire cell when EGF receptors were overexpressed or when receptor/ligand endocytosis was blocked. We thus present evidence that ligand-independent propagation of EGF signaling occurs only when the receptor density on the plasma membrane is high, such as in carcinoma cells. Introduction Signaling by ligand-activated receptor tyrosine kinases (RTKs), such as EGF receptor (EGFR), elicits a wide range of cell type-specific responses leading to proliferation, differentiation, apoptosis, and migration (reviewed in Hackel et al., 1999; Schlessinger, 2000). Upon ligand binding, RTKs dimerize and undergo autophosphorylation at specific tyrosine residues. In turn, these phosphotyrosines and their adjacent sequences specifically recruit signaling molecules via SH2 and PTB domains. The 5
Correspondence: [email protected]
recruited molecules are adaptor proteins, such as Shc, Grb2, and CrkII, or enzymes, like PLC␥. They mediate subsequent activation of a variety of downstream signaling cascades. Indicators for various cellular events have been developed by a green fluorescent protein (GFP)-based fluorescence resonance energy transfer (FRET) technique (Miyawaki et al., 1997; Janetopoulos et al., 2001; Ting et al., 2001; Zaccolo et al., 2000; Zhang et al., 2001; van Roessel and Brand, 2002). We also generated novel fluorescent indicators to monitor activation of Ras family G proteins (Raichu-Ras) (Mochizuki et al., 2001) and tyrosine phosphorylation (Picchu-X) (Kurokawa et al., 2001). Raichu-Ras consists of H-Ras, the Ras binding domain (RBD) of Raf, and yellow- and cyan-emitting mutants of GFP, YFP, and CFP, respectively. Intramolecular interactions between active, GTP-bound Ras and the RBD bring YFP and CFP into close proximity, thereby increasing FRET. Picchu-X consists of CrkII sandwiched with YFP and CFP. Intramolecular binding of the SH2 domain to a phosphorylated tyrosine increases FRET. Using these probes, we were able to obtain spatiotemporal images of EGF-induced Ras activation and tyrosine phosphorylation in single living cells. Upon application of EGF to cultured COS cells, which brings about global activation of EGFRs, Ras activation was observed to occur at the peripheral plasma membrane and was reduced at sites of cell-cell contact (Mochizuki et al., 2001), while tyrosine phosphorylation of CrkII beneath the plasma membrane was distributed evenly throughout the cell (Kurokawa et al., 2001). In most cell-biological experiments, cultured cells are bathed in ligand solutions, whereas cells in intact tissues are stimulated locally in most physiological situations. It would be interesting to learn how EGF signaling spreads within a cell after local stimulation with EGF. While activation of EGFR is generally thought to activate the RasRaf-MEK-ERK pathway and thereby convey information to the nucleus, evidence has accumulated that EGF signaling is involved in many other spatially dynamic cellular processes, such as membrane trafficking (Barbieri et al., 1998), turnover of focal adhesions (Schlaepfer and Hunter, 1998), and cytoskeletal organization (Ho and Bretscher, 2001). Therefore, it is important to understand whether the EGF signaling remains localized or is propagated in response to local stimulation. Verveer et al. (2000) reported a new signaling mechanism described as ligand-independent lateral propagation of receptor phosphorylation in the plasma membrane and suggested a dynamic equilibrium between monomeric and dimeric activated EGFR. The authors elegantly monitored EGFR phosphorylation by measuring FRET between GFP fused to the carboxyl terminus of the receptor and Cy3 attached to a phosphotyrosinespecific antibody. They observed rapid and extensive propagation of receptor phosphorylation over the entire cell (MCF7 cell) after local stimulation with EGF covalently attached to beads. In order to observe these cellular events, the investigators needed to introduce a large number of EGFRs into the cells, though this approach
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Figure 1. Visualization of Rhodamine-EGFEGFR Complexes Time-lapsed images of A431 cells (A and B), normal COS cells (C and D), and COS cells transfected with the cDNA for EGFR (E and F) when incubated with 50 ng/ml rhodaminelabeled EGF. The fluorescence signals were monitored every 10 s through the rhodamine channel. Images taken after 10 s (A, C, and E) and 20 min (B, D, and F) of incubation are presented. For estimation of EGFR plasma membrane expression level, images (C) and (E) were taken under the same optical conditions and are displayed using the same grayscale. In contrast, image (A) was captured with 20-fold-less sensitivity. (E) EGFR-overproducing COS cells and untransfected COS cells are indicated by asterisks and arrowheads, respectively. Scale bar, 10 m.
is known to make the observed cells cancerous. In such pathological situations, we also observed that local stimulation with EGF caused rapid and global propagation of Ras activation and tyrosine phosphorylation beneath the plasma membrane. Though the ligand-independent propagation mechanism initially proposed was understood to be at work in all cell types, we have argued against generalizing the results of the aforementioned experiments to suggest that this is a common physiological mechanism. In fact, here, we offer the first evidence that EGF signaling remains localized in cells that express EGFR at a basal level. A more comprehensive view of EGF signaling pattern must include the understanding that EGF-independent signal propagation depends on the density of EGFRs in the plasma membrane, which is, in turn, related to the saturable endocytosis (Wiley, 1988) of EGF-EGFR complexes. Results Visualization of EGF-EGFR Complex Dynamics in Membranes We followed the fate of rhodamine-labeled EGF molecules that were applied over A431 (human epidermoid carcinoma cell line) and COS cells. The ligands were added in Hank’s Balanced Salt Solution (HBSS) medium at a final concentration of 50 ng/ml. Immediately after application (10 s), the A431 cells displayed heavy labeling of their plasma membranes (Figure 1A), suggesting
high receptor density on these cells. At later time points (20 min), the A431 cells were found to have ineffectively internalized this high level of occupied EGFR, apparently because of a limited amount of a specific component responsible for mediating EGF-induced receptor internalization (Wiley, 1988). While a punctate pattern of fluorescence consistent with internalization was observed, a significant amount of rhodamine-EGF remained on the plasma membrane (Figure 1B). It was also noted that the EGF-stimulation induced refractile changes and detachment from the substratum. COS cells were treated with rhodamine-EGF in the same manner, and fluorescence images (Figures 1C–1F) were captured. These images required a longer exposure time than did the A431 cell images because of the comparatively low receptor expression of the COS cells. The ligands were again observed to be bound to EGFRs on the plasma membrane (Figure 1C), though quantitative analysis indicated an approximately 50-fold difference in the absolute signal intensity between COS and A431 cells. The EGFR densities on A431 and COS cells were previously reported to be roughly 2 ⫻ 106 and 4 ⫻ 104 molecules per cell, respectively (Wiley, 1988; Livneh et al., 1986). Assuming that a linear correlation between plasma membrane labeling and the actual number of receptors exists, it would then be possible to estimate roughly the receptor density from the plasma membrane fluorescence intensity. By 20 min after stimulation, the fluorescent labeling on the plasma membrane of the
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COS cells had disappeared, and punctate signals had emerged at the periphery and in the perinuclear region (Figure 1D), indicating that most of the EGF-EGFR complexes had been internalized into endosomal compartments. EGFR internalization was also visualized in COS cells by immunocytochemical analysis with an antiEGFR antibody (data not shown). The resulting immunosignals displayed similar patterns to the rhodamineEGF signals. Thus, the rhodamine-EGF image can be viewed as roughly reflecting the distribution of EGFR, in spite of the segregation of ligand and receptor at late stages of endocytosis (Carter and Sorkin, 1998). When COS cells were transfected with EGFR cDNA, a portion of the cells produced a large quantity of receptors. Ten seconds after stimulation with rhodamine-EGF, the plasma membranes of the transfected cells (Figure 1E, asterisks) were intensely labeled. Later, these EGFRoverexpressing cells also displayed saturated EGFEGFR endocytosis (Figure 1F), as did the A431 cells from the previous experiment. The fluorescence-signal intensities of the cells in Figures 1C and 1E were comparable, since the two images were taken under the same conditions. The EGFR density of the transfected cells was estimated to be around 5 ⫻ 105 molecules per cell. Clearly, the cells indicated by arrowheads in Figure 1E were untransfected, as they exhibited the same rhodamine-EGF labeling as the control cells in Figures 1C and 1D. Local Stimulation by the Use of Laminar Flow Figure 2A is a diagram of the microfluidic channel that was used to subject cells to local stimulation (Takayama et al., 1999, 2001). It has three inlets and one outlet. The optimal channel size for our COS cell/EGF experiments was attained with 150 m-wide rectangular capillaries. The performance of the channel with confluent COS cells (Figure 2B) was examined with fluorescent EGF molecules. FITC- and rhodamine-labeled EGF were made to flow over the COS cells from the left and right inlets, respectively, together with medium from the middle inlet. A merged green and red fluorescence image is shown in Figure 2C. Stable laminar streams were always maintained for over one hour. We found that a submaximal dose of EGF gave rise to a graded amplitude of Raichu-Ras signal, while a maximal dose increased the amplitude to a saturated level. In culture dishes, cells are exposed to EGF ligands in the medium, and it is by this system that most previous studies regarding EGF signaling have been carried out. In our microfluidic channels, on the other hand, cells are continuously exposed to moving EGF ligands in the flow stream. For this reason, we decided to examine the relationships between EGF concentration and the amplitude of Raichu-Ras signal in normal COS cells using both experimental systems. The dose-response curve obtained with the channel was found to be shifted slightly to the right (Figure 2D). Local Activation of Ras in Normal COS Cells We analyzed the spatiotemporal pattern of Ras activation in response to a laminar flow in a normal COS cell plated in the microfluidic channel (Figure 3). The cell
that expressed Raichu-Ras protein was stimulated locally with a solution containing 50 ng/ml of rhodamineEGF. This concentration of EGF ligands should be saturating within the stimulated region (see Figure 2D). A fluorescence image of the cell was taken through the FRET channel (440 ⫾ 10 nm excitation, 535 ⫾ 12.5 nm emission) (Figure 3A). Rhodamine-EGF flowed over the right-hand section, as indicated by violet shading. To confirm the presence of a stable, controlled laminar flow, we monitored the red fluorescence of the bound rhodamine-EGF through the rhodamine channel (546 ⫾ 5 nm excitation, 595 ⫾ 30 nm emission). After stimulation for 10 min, the area to which the ligands bound was visualized, including the neighboring cells that did not express Raichu-Ras protein (Figure 3B). The focal plane was on the top plasma membrane, and, therefore, the punctate fluorescence pattern characteristic of endocytosis is not seen. Internalization of EGF-EGFR complexes, however, was visualized by viewing a different focal plane (data not shown). Figure 3D shows representative pseudocolored emission-ratio images of Raichu-Ras, which were made by assigning red and blue to high and low levels of Ras activation, respectively. In addition, the temporal profiles of the ratio values in the regions of interest (ROIs) indicated in Figure 3A are shown (Figure 3H). Ras activation increased and reached a maximum within 10 min in the region that was exposed to rhodamine-EGF. In contrast, little or no activation was seen in the unexposed parts of the cell. After 10 min, a noticeable similarity between the patterns of rhodamine-EGF binding (Figure 3B) and Ras activation (Figure 3D) was observed. The outlines of the cell at 0 and 20 min are superimposed (Figure 3C). Although this COS cell was surrounded by cells on every side, the spatially restricted stimulation resulted in a slight, but definite, morphological change that was localized to the stimulated region. Such morphological change occasionally affected the flow pattern, resulting in an indistinct leak of EGF ligands. Thus, Raichu-Ras signal was observed to ooze slightly out of the stimulated region (Figure 3D). But the leaking signal was only transient and never crossed the cell at a later time point (⬎1 hr). Another normal COS cell did not show any morphological change with the rhodamine-EGF flow over almost half the area of the cell (Figures 3E and 3F). The Ras signal reached the maximum at 10 min and then gradually faded out (Figures 3G and 3I). Similar experiments were performed using 11 different normal COS cells. In all cases, local stimulation with EGF resulted in localized activation of Ras (Table 1). Localized Tyrosine Phosphorylation in Normal COS Cells Next we observed tyrosine phosphorylation events in a normal COS cell expressing Picchu-X (Figure 4A), which, again, employs FRET between YFP and CFP (Kurokawa et al., 2001). This in vivo probe has the CAAX box of KiRas at its carboxyl terminus. This modification restricts its localization to the plasma membrane, thereby improving the sensitivity and spatial resolution of the system for detecting tyrosine phosphorylation. This plasma membrane localization also suggests that the Picchu-X signal may reflect the phosphorylation status of EGFR. In fact, the requirement of EGFR kinase activity for the
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Figure 2. Microfluidic Channel for Laminar Flow Experiments (A) Top view of the capillary network. (B) A bright-field micrograph showing the junction where the three inlets converge. The channel contained confluent COS cells. A 20⫻ objective lens was used. (C) Fluorescence micrograph of the same field as in (B). The FITC-EGF and rhodamineEGF fluorescence images were captured separately and merged. Scale bar, 100 m. (D) Dose-response curves for EGF-induced Ras activation in normal COS cells expressing Raichu-Ras. The EGF-induced increase in the emission ratio of Raichu-Ras (R/R0; the relative difference of the observed ratio [R] and the prestimulus value [R0]) was measured in culture dish (dotted line) and in the microfluidic channel (solid line). The saturating level of R/R0 ranged from 1.2 to 1.4. Each point is the mean ⫾ SD of three to six experiments.
Picchu-X signal was confirmed pharmacologically (Kurokawa et al., 2001). Consistent with the view that Picchu-X is an early detector of EGF signaling, its emission ratio started to increase immediately and reached its maximum within 1 min after the addition of EGF (Figure 4D). Like the Ras activation shown in Figure 3, tyrosine phosphorylation signals were localized to the same portion of the cell as the EGF stimulation (Figures 4C and 4D), where rhodamine-EGF was bound (Figure 4B). There was no observable tyrosine phosphorylation signal in the left part containing ROIs 3 and 4 , even after 25 min of stimulation. Subsequently, 50 ng/ml rhodamineEGF were added to the left stream to stimulate the nonresponding region. An immediate increase in the emission ratio was observed (Figure 4E), indicating that the EGFRs in the left region had not been desensitized. Localized Versus Propagated EGF Signaling Our results mentioned above seemed to contradict the previous report by Verveer et al. (2000), which concluded that the phosphorylation of EGFR was propagated quickly after focal stimulation with EGF beads. There were, however, a couple of differences between the two experimental protocols. In their experiments, first, EGFR-GFP chimeric proteins were highly expressed in MCF7 carcinoma cells to allow monitoring the phosphorylation of the exogenous EGFR. The density of
EGFRs on the cells would have then been remarkably high (5 ⫻ 105 per cell) compared with normal COS cells. Second, the cells were focally stimulated using 0.8 mdiameter beads conjugated with EGF molecules. Although the ability of these beads to activate EGFR and the subsequent internalization of the receptor-ligand complexes have been shown (Verveer et al., 2000; Brock and Jovin, 2001), artifacts due to this mode of EGF delivery cannot be disregarded. We hypothesized that the dynamics of EGFR activation could depend on the density of receptors in the plasma membrane. To test this hypothesis, we increased the receptor density by expressing EGFR in normal COS cells. Also, since endocytosis of ligandreceptor complexes is closely related to the receptor density and is important for regulation of signaling, we opted to use the soluble form EGF to allow physiological endocytosis to occur. Spatial Propagation of EGF Signaling in Cells Overexpressing EGFR COS cells in the microfluidic channel were cotransfected with cDNAs for Raichu-Ras and full-length EGFR. EGFR and Raichu-Ras were expressed with various ratios in the transfected cells. The Raichu-Ras-expressing cell shown in Figure 5A expressed a significant amount of EGFR. This cell was next to another that displayed a
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Figure 3. Local Stimulation of Normal COS Cells with Rhodamine-EGF for Visualization of Ras Activation (A) Fluorescence image of Raichu-Ras before stimulation. The region of rhodamine-EGF exposure is shaded in violet. Across the laminar flows, four ROIs are assigned. Scale bar, 10 m. (B) Rhodamine-EGF fluorescence after 10 min stimulation. The outline of the COS cell is shown by a dotted line. (C) Superimposed outlines of the cell at 0 (black) and 20 (red) min. (D) A series of dual-emission ratio (535 ⫾ 12.5 nm to 480 ⫾ 15 nm) images presented in intensity-modified display (IMD) mode. Time points (min) after initiation of stimulation are shown in the top right corners. (E) Fluorescence image of Raichu-Ras in another normal COS cell before stimulation with rhodamine-EGF (violet). Four ROIs are assigned as in (A). Scale bar, 10 m. (F) Rhodamine-EGF fluorescence after 20 min stimulation in the COS cell shown in (E), which is outlined by a dotted line. The lens was focused to see the punctate fluorescence pattern characteristic of endocytosis. (G) A series of dual-emission ratio images from the COS cell shown in (E), presented in a manner similar to that in (D). (H and I) Temporal profiles of the ratios in the ROIs indicated in (A) and (E), respectively. The changes in ratio were normalized to the initial ratio values. Red circles and squares are the data points from the stimulated side: ROIs 1 and 2, respectively. Blue circles and squares are from the unstimulated side: ROIs 3 and 4, respectively.
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Table 1. EGFR Expression Dependency of EGF Signaling Propagation Indicators
EGF Binding Sites/Cell
Raichu-Ras
3–5 ⫻ 104 3–5 ⫻ 104
⫹EGFR
Picchu-X ⫹EGFR
Propagation/Number of Experiments ⫹MDC
1–3 ⫻ 105 3–6 ⫻ 105 6–8 ⫻ 105 3–5 ⫻ 104 3–5 ⫻ 104 1–3 ⫻ 105 3–6 ⫻ 105
0/13 3/3 3/3 4/4 2/2
⫹MDC
0/8 2/2 1/1 4/4
The results of 40 experiments are summarized. In every experiment, the observed cells were mostly confluent and were stimulated over 10–50% of their surface areas.
similar level of EGFR expression, yet did not express Raichu-Ras (Figure 5B, arrowhead). The arrow in Figure 5B indicates the presence of cells that produced neither Raichu-Ras nor exogenous EGFR; the fluorescence signal from the rhodamine-EGF on untransfected COS cells was barely visible because of the short exposure time optimized for the high levels of EGFR on the transfected cells. From the intensity of the red plasma membrane fluorescence, the cell of interest was estimated to contain roughly 4 ⫻ 105 binding sites. After 1 min of local stimulation, the Raichu-Ras signal was noticeable across the entire cell and reached a maximum within 10 min in both the stimulated and unstimulated regions (Figures 5C and 5G). It should be noted that the majority of rhodamine-EGF-EGFR complexes remained in the plasma membrane because of saturation of endocytosis (Figures 1E, 1F, and 5B). Such global activation of Ras was observed in each of eight other COS cells that overexpressed EGFR in a range from 1 ⫻ 105 to 8 ⫻ 105 molecules per cell (Table 1). Next, another COS cell overexpressing EGFR (8 ⫻ 105 binding sites) was locally stimulated with a submaximal dose of rhodamine-EGF (1 ng/ml) (Figures 5D and 5E). The Raichu-Ras signal increased to a submaximal level, evenly throughout the cell (Figures 5F and 5H). The subsequent application of 50 ng/ml rhodamine-EGF then caused a further increase in the signal to a saturating level (Figure 5H). The ligand-independent propagation of EGFR phosphorylation (or activation) after focal stimulation (Verveer et al., 2000) was substantially reproduced in our experiment that employed a COS cell expressing Picchu-X and a high number of EGFRs (3 ⫻ 105 binding sites per cell) (Figure 6). Figures 6C and 6D show that local stimulation (Figures 6A and 6B) for 3 min resulted in a global increase in the emission ratio of Picchu-X, suggesting full activation of EGFRs throughout the cell. It should be noted that we observed this spread of tyrosine phosphorylation events in a single live COS cell in real time, while the previous study used many different cells that were fixed at various time points and reacted with anti-phosphotyrosine antibody. Mechanisms for Localized EGF (Ras) Signaling We studied further the mechanisms underlying the localized activation of Ras in normal COS cells, as presented
in Figure 3. First, we examined whether endocytosis of EGF-EGFR complexes negatively regulated the propagation of Ras activation. COS cells in the microfluidic channel were pretreated with 20 M monodansylcadaverine (MDC), an inhibitor of clathrin-dependent endocytosis, for 10 min. Then, 50 ng/ml of rhodamine-EGF were applied to the left part of a COS cell expressing RaichuRas (Figure 7A), whereupon the plasma membrane on that side became homogeneously labeled, though some bright spots were also detected (Figure 7B). No punctate fluorescence pattern was observed in different focal planes. The surface labeling lasted for over 20 min, indicating that the blockade of ligand-receptor endocytosis had occurred. The resulting accumulation of EGF-EGFR complexes in the plasma membrane resulted in gradual, but global, activation of Ras within the entire cell (Figure 7C). The emission ratio of Raichu-Ras increased by about 40% after 20 min. Importantly, this Ras activation signal was specifically due to the EGF stimulation. In comparison with the strong Ras signal observed in the tested cell, the increase in the emission ratio in adjacent cells (Figure 7C, arrowheads), which had also been treated with MDC, was negligible, at only 1% of the basal level. Gradual propagation of tyrosine phosphorylation was observed also in Picchu-X-expressing COS cells that had been treated with MDC (Table 1). These results support the idea that EGFRs in normal COS cells are effectively downregulated by endocytosis to prevent lateral propagation of EGF signaling. Similar global delocalization of Ras activation and tyrosine phosphorylation was observed in normal COS cells expressing a moderate amount of an oncogenic version of c-Cbl (70ZCbl) (Levkowitz et al., 1999; Lill et al., 2000; Soubeyran et al., 2002), which recent results argue prevents endocytosis. Next, we examined the activation of another Ras family G protein, Rap1, which is activated inside cells by EGF and in a manner dependent on endocytosis. A COS cell expressing Raichu-Rap1 (Mochizuki et al., 2001) was locally stimulated in the microfluidic channel (Figure 7D). After 20 min of stimulation, EGF-EGFR complexes were mostly internalized, but localized within the stimulated region (Figure 7E). Activation of Rap1, however, was initiated in the perinuclear region and spread in an isotropic manner toward the plasma membrane (Figure
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Figure 4. Local Stimulation of a Normal COS Cell with Rhodamine-EGF for Visualization of Tyrosine Phosphorylation beneath the Plasma Membrane (A) Fluorescence image of Picchu-X with four ROIs; the rhodamine-EGF flow is shaded in violet. Scale bar, 10 m. (B) Rhodamine-EGF fluorescence images after 10 and 20 min stimulation, with the outline of the COS cell depicted by a dotted line. Over the monolayer culture is a round cell, which was intensely labeled (indicated by an asterisk). (C) A series of dual-emission ratio images presented in IMD mode. Time points (min) after the start of stimulation are shown in the top right corners. (D) Temporal profiles of the ratios in the ROIs indicated in (A). Data points are plotted as in Figures 3H and 3I. (E) At 30 min, a ratio image was captured after global stimulation with rhodamine-EGF.
7F). This observation suggests that the guanine nucleotide exchange factor for Rap1 may be activated not only by the internalized EGFRs, but also by diffusible messenger molecules, and that the activation of Rap 1 outside the stimulated region may block the propagation of Ras-dependent pathways inside the cells. It is interesting that activation of Ras, but not Rap1, retains the spatial information regarding the source of EGF signaling in the plasma membrane. Discussion When a cell is stimulated locally with ligands, is the activation of intracellular signaling localized or propagated over the entire cell? To address this intriguing and fundamental question, we investigated the spatiotemporal pattern of EGF signaling in single live COS
cells. A summary of our results is presented in Table 1. We demonstrate that ligand-independent lateral signal propagation depends on the plasma membrane receptor density, helping to explain the previous findings reported on this subject. To obtain a better understanding of the regulation of signaling dynamics, we also employed the following three technologies recently developed in our laboratories: (1) Raichu-Ras (Mochizuki et al., 2001) and Picchu-X (Kurokawa et al., 2001), fluorescent indicators that can pinpoint the site of Ras activation and tyrosine phosphorylation beneath the plasma membrane, respectively, within single live cells; (2) microfluidic systems, which create controlled, stable laminar flow to make possible finely localized treatment of cells with soluble ligands (Takayama et al., 1999, 2001); (3) an advanced multicolor imaging system for conventional fluorescence microscopy (Sawano et al., 2002).
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Figure 5. Local Stimulation of EGFR-Overproducing COS Cells with Rhodamine-EGF for Visualization of Ras Activation (A) Fluorescence image of Raichu-Ras in a COS cell transfected with EGFR cDNA. The cell was nearly in contact with the left wall of the capillary. Four ROIs and the rhodamine-EGF flow (violet) are shown. Scale bar, 10 m. (B) Rhodamine-EGF fluorescence after 10 min stimulation. The dotted line depicts the outline of the COS cell. A neighboring cell, which produced a large quantity of EGFRs, is indicated by an arrowhead. An arrow defines other neighboring cells that were not transfected. This image was taken with 10-fold-less sensitivity than the images in Figures 3B and 4B. (C) A series of dual-emission ratio images presented in IMD mode. Time points (min) after initiation of stimulation are shown in the top right corners. (D) Fluorescence image of Raichu-Ras in another EGFR-overexpressing COS cell before stimulation with rhodamine-EGF (violet). Four ROIs are assigned as in (A). Scale bar, 10 m. (E) Rhodamine-EGF fluorescence after 10 min stimulation in the COS cell shown in (D), which is outlined by a dotted line. (F) A series of dual-emission ratio images from the COS cell shown in (D), presented in a manner similar to that in (C). (G and H) Temporal profiles of the ratios in the ROIs indicated in (A) and (D), respectively. Data points are plotted as in Figures 3H and 3I. Subsequent addition of 50 ng/ml rhodamine-EGF is indicated by an arrowhead in (H).
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Figure 6. Local Stimulation of an EGFROverproducing COS Cell with RhodamineEGF for Visualization of Tyrosine Phosphorylation beneath the Plasma Membrane (A) Fluorescence image of Picchu-X with four ROIs, with the rhodamine-EGF flow shaded in violet. Scale bar, 10 m. (B) Rhodamine-EGF fluorescence images after 10 and 20 min stimulation. The outline of the COS cell is demarcated by a dotted line. The camera settings were the same as those for the image in Figure 5B. (C) A series of dual-emission ratio images presented in IMD mode. Time points (min) after initiation of stimulation are shown in the top right corners. (D) Temporal profiles of the ratios in the ROIs indicated in (A). Data points are plotted as in Figures 3H and 3I.
Previous studies of EGF signaling have been performed using cells that express high levels of EGFRs. In one study, GFP variants were fused to the carboxyl terminus of the EGFR. The functionality of the EGFRGFP was verified (Carter and Sorkin, 1998), and the proteins were used to monitor phosphorylation (Verveer et al., 2000) and endocytosis of the EGFR (Carter and Sorkin, 1998) and the interaction between the EGFR and adaptor proteins (Sorkin et al., 2000). Expression of EGFR-GFP, however, results in a high density of functional receptors on the plasma membrane, creating a situation where receptor activation spreads via dynamic dissociation-association equilibrium, as proposed by Verveer et al. (2000). Therefore, we avoided the introduction of any GFP-labeled EGFR as an indicator. Instead, we focused on visualizing two EGF-dependent events: Ras activation and tyrosine phosphorylation beneath the plasma membrane. Another study used A431 carcinoma cells, which have an extraordinarily high number of EGFR molecules (1–3 ⫻ 106 per cell) (Wiley, 1988). It is reasonable to assume that, according to the above
argument, EGFR dimers (oligomers) are formed before the 1:1 interaction with EGF, as revealed by single-molecule imaging (Sako et al., 2000) or FRET analysis (Gadella and Jovin, 1995). However, because of autocrine regulation of A431 cells through production of transforming growth factor ␣ (TGF-␣) (Harder et al., 1998), it would be difficult to attribute changes to local stimulation with EGF. Also, because the cells exhibited refractile changes and detachment from the substratum upon EGF treatment (Figures 1A and 1B), we concluded that these cells were inappropriate for our imaging experiments with laminar flows. As a result, we chose to use COS cells, which usually express a small number of EGFRs (3–5 ⫻ 104 per cell) (Livneh et al., 1986). Overexpression of cDNA-derived EGFR greatly increased the receptor density on the surface of the cells. Transfected cells that displayed a moderately high number of EGFRs (1–8 ⫻ 105 per cell) and that were adhesive to the substratum were used for imaging experiments. A comparative study using normal and transfected COS cells was performed to give us a
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Figure 7. Propagation of Ras Activation after Blockade of Endocytosis and Visualization of Rap1 Activation in Normal COS Cells (A) Fluorescence image of Raichu-Ras in a COS cell after treatment with MDC but before stimulation. This cell was neighbored by a few other cells on the right side, as indicated by arrowheads (C). The rhodamine-EGF flow (violet) is shown. Scale bar, 10 m. (B) Rhodamine-EGF fluorescence images after 10 and 20 min stimulation. The outline of the COS cell is shown by a dotted line. (C) A series of dual-emission ratio images for Ras activation presented in IMD mode. Time points (min) after initiation of stimulation are shown in the top right corners. After 20 min, no activation of Ras was observed in the neighboring cells (arrowheads), which had also been treated with MDC. (D) Fluorescence image of Raichu-Rap1 in a COS cell with the rhodamine-EGF flow shaded in violet. Scale bar, 10 m. (E) Rhodamine-EGF fluorescence images captured after 10 and 20 min stimulation. The outline of the COS cell is shown by a dotted line. (F) A series of dual-emission ratio images of Rap1 activation presented in IMD mode. Time points (min) after initiation of stimulation are shown in the top right corners.
direct answer to the question of how EGFR density affects the propagation of receptor activation and its downstream events. The central finding in our study is that, in normal COS cells, local stimulation with a maximal dose (50 ng/ml) of rhodamine-EGF led to spatially restricted EGF signaling. It should be noted that the saturable activation of EGFR in a local region effectively confines the downstream signaling. We previously reported the sustained activation of Ras for over 10 hr in the neurites of PC12 cells expressing Raichu-Ras (Mochizuki et al., 2001). Since the fluorescence intensity of PC12 neurites expressing YFPRas recovered within 4 min after photobleaching, it was concluded that the local activity of Ras is determined by a local balance between the activity of guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP), rather than by the retention of activated Raichu-Ras (Mochizuki et al., 2001). The localization of the
signals of Raichu-Ras and Picchu-X in the present study also suggests that the indicators have rapid kinetics of activation and deactivation. The internalization of activated receptors is generally viewed as a mechanism to attenuate signaling. Activation of EGFRs by ligands also triggers rapid endocytosis of EGF-EGFR complexes. Although it has been reported that certain pathways mediated by EGFR are activated in endosomes (Sorkin and Carpenter, 1991; Haugh et al., 1999), the internalized EGFR is mostly ubiquitinated and then degraded in proteosomes (Waterman et al., 2002). This process is controlled by complex formation of Cbl with CIN85-endophillin (Soubeyran et al., 2002; Petrelli et al., 2002). Another recent study has shown that the endocytosed EGFR is efficiently inactivated through dephosphorylation by protein tyrosine phosphatase (PTP) 1B on the surface of the endoplasmic reticulum before degradation (Haj et al., 2002). In either case, the
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endocytosis plays a pivotal role in the inactivation of EGFR. Although small beads conjugated with EGF molecules have often been used for focal stimulation and have been observed to be taken up by cells (Verveer et al., 2000; Brock and Jovin, 2001), they may impede normal physiological internalization and metabolism of the EGF-EGFR complexes. Thus, we studied the internalization of ligand-receptor complexes using rhodaminelabeled EGF for stimulation. The pH-resistant fluorescence of rhodamine permitted quantitative monitoring in acidic organelles, such as endosomes, though segregation of ligand and receptor does occur at these late stages. We reproduced the results of Figures 3–7 using unlabeled EGF (data not shown), to prove that rhodamine conjugation does not affect its interaction with EGFRs. How is endocytosis involved in the spatiotemporal pattern of EGF signaling that we observed in this study? When EGFR-overexpressing COS cells were stimulated, a large number of EGFRs remained in the membrane well after stimulation, most likely due to saturation of EGF-EGFR endocytosis. This situation probably facilitated transient dimer formation of EGFR in the plasma membrane, resulting in the observed ligand-independent propagation of receptor activation. In normal COS cells, by contrast, the EGFRs are efficiently cleared off the plasma membrane soon after stimulation, allowing for localized receptor activation. Thus, when EGF-EGFR endocytosis was blocked, the receptor activation was gradually delocalized. Within a cell, on the other hand, diffusible factors may work to augment the propagation of receptor activation. Recent evidence has shown that activation of EGFR results in the generation of hydrogen peroxide (H2O2), which upregulates tyrosine phosphorylation signaling by inhibiting PTP activity (Rhee et al., 2000). It is unlikely that the internalized activated EGFRs contributed to the Raichu-Ras and Picchu-X signals observed in our study because the activation of both indicators is exclusively localized to the plasma membrane (Mochizuki et al., 2001; Kurokawa et al., 2001). Even if the internalized EGFRs act on the nearby plasma membrane, they are most likely not involved in the lateral propagation of EGF signaling shown in Figures 5 and 6. When EGFR was overexpressed, internalization of the ligand-receptor complexes was localized to the stimulated side, and the red fluorescence did not spread to the opposite side of the cell (see Figure 6B). There are many interesting biological consequences of the observed local and global activation of EGF signaling. One of the well-studied downstream signaling targets of EGFR is the Ras-Raf-MEK-ERK pathway. Since this pathway converges on the nucleus, the spatial information pertaining to the site of EGFR activation is likely to be lost. In contrast, other EGFR-dependent pathways, such as membrane trafficking, turnover of focal adhesions, and cytoskeleton organization, are spatially restricted, contributing to directional cell morphology and motility. In our experiments, the localized spatial pattern of EGF signaling was well correlated with local morphological changes (Figure 3C), potentially involving not only the Ras pathway, but also other EGF-induced signaling molecules, such as Rho, focal adhesion kinase (Lu et al., 2001), and phosphatidylinositol-3 kinase (Arcaro et al., 2000). In organogenesis, local activation of
EGF signaling appears to be important for coordinated control of region-specific detachment from the extracellular matrix. This process may allow for proliferation of cells with regulated EGFR expression while maintaining architectural order. On the other hand, a variety of carcinoma cells overexpress EGFR and show dysregulated cell motility upon EGF treatment, as do A431 cells. Ligand-independent propagation of EGF signaling may be one of the molecular mechanisms underlying invasion and metastasis, criteria by which malignant tumors are characterized. Experimental Procedures Microfluidic Channels Polydimethylsiloxane (PDMS) slabs with channel features were made as previously described (Takayama et al., 1999). Capillary channels were formed by placing the PDMS membrane on a cover glass that had been coated with 0.25% Poly-L-Lysine (Sigma) for 5 hr. Rhodamine-EGF and FITC-EGF were purchased from Molecular Probes. Cell Cultures COS cells and A431 carcinoma cells were maintained in Dulbecco’s modified eagle’s medium (DMEM) containing 10% fetal bovine serum, antibiotics, and glutamine (growth medium) in a 5% CO2 incubator at 37⬚C. For delivery of COS cells into the capillary channels, the outlet reservoir was filled with a suspension of cells (about 105 cells per milliliter in the growth medium). The cells were allowed to flow slowly into the main channel and adhere to the cover glass. Nonadherent cells were removed by washing. The cells were then incubated in 5% CO2 at 37⬚C for 1 day before cDNA transfection. Transfection Cells were transfected with pRaichu-Ras (Mochizuki et al., 2001), pPicchu-X (Kurokawa et al., 2001), pRaichu-Rap1 (Mochizuki et al., 2001), or pKU-EGFR (Gotoh et al., 1997) for expression of EGFR, by the Lipofectin method (Gibco BRL). COS cells in the channel were treated with the transfection solution in the presence of 5% CO2 at 37⬚C for 16 hr. After washing, the capillary system was filled with growth medium and incubated for another 1 or 2 days. Imaging For imaging experiments, the growth medium in the capillary system was replaced by HBSS buffer. Cells were imaged on an inverted microscope (Olympus IX70) with a standard 75 W xenon lamp, a 40⫻ objective lens (Uapo/340, N.A. 1.35), and a dichroic mirror (455 nm long pass). Interference filters (excitation and emission filters) set in wheels were mechanically automated using Lambda 10-2 hardware (Sutter Instruments, Novato, CA). Emitted light was captured with a cooled CCD camera (Cool Snap fx; Roper Scientific, Tucson, AZ). The whole system was controlled using MetaFluor 4.6.6 software (Universal Imaging, Media, PA). Raichu and Picchu-X both have cyan and yellow fluorescent proteins as donor and acceptor, respectively. For dual-emission ratio imaging, fluorescence pictures were captured alternately through the donor channel (440 ⫾ 10 nm excitation, 480 ⫾ 15 nm emission) and FRET channel (440 ⫾ 10 nm excitation, 535 ⫾ 12.5 nm emission). Rhodamine-EGF was imaged through the rhodamine channel (546 ⫾ 5 nm excitation, 595 ⫾ 30 nm emission). All fluorescence images were taken using the 455 nm-long pass dichroic mirror. About 5% of the total light at 546 nm was reflected by the dichroic mirror and was used for excitation of rhodamine (Sawano et al., 2002). Through the donor and FRET channel, transfected cells that expressed a moderate amount of the fluorescent indicator and were located close to the capillary junction were selected. A solution of rhodamine-EGF was then substituted into either the left or right inlet, so that the solution flowed over one of the edges of the selected cell. For blocking of endocytosis, the three inlets were filled with HBSS buffer containing 20 M monodansylcadaverine (MDC) (Calbiochem) for 10 min.
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