How intracellular signals are channeled across cells Laboratory for Cell Function Dynamics 1. Background When I had no clear answer to how a signal occurring from local cell stimulation is transferred to other regions, a new view suggested that "signaling raised by local stimulation quickly propagates throughout the cell" through lateral propagation that involves transient receptor dimers (Verveer, P. J. (2000) Science 290 1567). Although they claimed that signaling propagation independent of stimulation occurred in experiments, skepticism remained. In normal cells, actions that occur following local stimulation, such as cell proliferation or differentiation, are spatially controlled in an elaborate manner ; therefore, I was reluctant to believe that signal propagation occurs according to its own properties regardless of the spatial characteristics of the stimulation. The authors of the above mentioned article over-expressed EGFR-GFP (epidermal growth factor receptor fused with GFP) in cells to visualize the phosphorylation signal, which created a cancer-like state in the cell. To make local stimulation possible, 0.8É m beads coated with EGF (epidermal growth factor) were made as stimulation targets. I had misgivings that the naturally negative accommodation mechanism-whereby the EGF-EGFR complex is endocytosed and metabolized-may be suppressed by these beads. To answer the basic question of whether signaling after local stimulation "stays within the stimulated site" or "propagates throughout the cell" we developed a new method. 2. How to visualize EGF signaling? External stimulation received by receptors on cell membrane issues a specific signal through interactions with various proteins and plays a role in the expression of cell proliferation or differentiation while forming a characteristic spatio-temporal pattern. To visualize this, we used 1) two types of fluorescent indicator (probe) to sense intracellular signaling propagation, and 2) a device to apply local stimulation on cells. Then we analyzed how signaling propagates from a spatio-temporal context. In cooperation with Dr. Michiyuki Matsuda at the Osaka University Microorganism Institute, we visualized local intracellular signaling, using a fluorescent probe (Picchu-X) to see EGFR's autophosphorylation, which occurs immediately after EGF colligation, and another fluorescent probe (Raichu-Ras) to capture Ras protein activation downstream. These probes did not affect the natural signaling propagation system of a cell, even when a sufficient expression for imaging was induced. Working collaboratively with Dr. Shuichi Takayama at Michigan University, we applied hydrodynamics technology (PARTCELL) to cause EGF local stimulation on cells (see Figures A, B and C). First, we developed an environment where a single cell would be exposed simultaneously to two flows : one containing EGF and another that did not containing EGF in laminar flow, which we created by making a narrow flow path and a stream at low speed. EGF labeled with Rhodamine (Rhodamine-EGF) remains water-soluble in acting with cells. Hence, unlike the bead experiment, it is possible to induce receptor inactivation along the endocytosis path following receptor complex formation. 3. Determining the spatio-temporal pattern of EGF signaling We plated normal COS cells (EGFR = 40.000/cell) on a PARTCELL cover glass and caused Picchu or Raichu-Ras expression (Figure D, upper left). After stimulating with Rhodamine-EGF on one side of a single cell (Figure D, upper-right white part), we observed that the tyrosine phosphorylation signal and Ras activation stayed within the stimulated site (Figure D, lower: parts that changed from blue to red indicate the activated site). We next over-expressed the EGF receptor in COS cells to increase the EGFR density (EGFR = 500.000/cell) on the plasmamembrane. As a result, both of these phenomena propagated across cells quickly following stimulation (Figure E). These results indicated that an intracellular signaling pattern after EGF stimulation was dependent on the EGFR density on the cell membrane. In addition, when normal COS cells were pre-treated with an inhibitor of endocytosis, activated EGFR accumulated in the plasmamembrane resulting in gradual signal propagation across cells after local stimulation. These results demonstrate that the receptor density on plasmamembrane and its ability to draw in activated receptors (endocytosis) were the decisive factors in signal behavior: i.e. would it "stay within the stimulated site" or would it "propagate throughout the cell". Armed with this information, it is possible to predict the spatio-temporal pattern that the tyrosine phosphorylation signal would propagate when a cell receives local EGF stimulation. In many experiments conducted in the past, as researchers took almost no account or control of these parameters, and we were not able to gain a comprehensive understanding from those experiment data. For example, our findings indicate that, when introducing a large amount of EGFR-GFP into cells as many researchers do, tyrosine phosphorylation signal localization may not be observed depending on the cell. The results of this research provide a clearer answer to this question and a good opportunity to review the existing research data. 4. Future prospects This series of experiments, which took into consideration the actual status of an organism, is expected to provide important knowledge in analyzing physiological organogenic kinetics in the developing process. In research of the cerebral nerve system in particular, in which the forms of individual cells are extremely diversified, it is important to analyze signaling propagation after local stimulation and, hence, these research findings will make great contributions to the study of brain science. Furthermore, the results will enable us to understand the mechanism of pathological phenomena of cell proliferation, such as infiltration or metastasis of cancer cells, as well as understand the suppression mechanism. In other words, it offers a new approach to suppress the aberrant proliferation of cancer cells by controlling the receptor density on plasmamembrane by accelerating the endocytosis ability of oncogenic receptors. In this vein, it is also expected to have a great impact on clinical medicine. Sawano A, Takayama S, Matsuda M, Miyawaki A (2002). Lateral propagation of EGF signaling after local stimulation is dependent on receptor density. Dev. Cell. 3. 245-257.